Blood Plasma Fractions as a Treatment for Aging-Associated Cognitive Disorders

ABSTRACT

Methods and compositions for treating and/or preventing aging-related conditions are described. The compositions used in the methods include fractions derived from blood plasma with efficacy in treating and/or preventing aging-related conditions such as neurocognitive disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application No. 62/412,258, filedOct. 24, 2016; the disclosure of which application is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention pertains to the prevention and treatment ofaging-associated disease. The invention relates to the use of bloodproducts, such as blood plasma fractions, to treat and/or preventconditions associated with aging, such as neurocognitive andneurodegenerative disorders.

BACKGROUND

The following is offered as background information only and is notadmitted to be prior art to the present invention.

Aging is an important risk factor for multiple human diseases includingcognitive impairment, cancer, arthritis, vision loss, osteoporosis,diabetes, cardiovascular disease, and stroke. In addition to normalsynapse loss during natural aging, synapse loss is an early pathologicalevent common to many neurodegenerative conditions, and is the bestcorrelate to the neuronal and cognitive impairment associated with theseconditions. As such, aging remains the single most dominant risk factorfor dementia-related neurodegenerative diseases such as Alzheimer'sdisease (AD) (Bishop, N. A. et al., Neural mechanisms of ageing andcognitive decline. Nature 464(7288), 529-535 (2010); Heeden, T. et al.,Insights into the ageing mind: a view from cognitive neuroscience. Nat.Rev. Neurosci. 5(2), 87-96 (2004); Mattson, M. P., et al., Ageing andneuronal vulnerability. Nat. Rev. Neurosci. 7(4), 278-294 (2006)). Agingaffects all tissues and functions of the body including the centralnervous system, and a decline in functions such as cognition, canseverely impact quality of life. Treatment for cognitive decline andneurodegenerative disorders has had limited success in preventing andreversing impairment. It is therefore important to identify newtreatments for maintaining cognitive integrity by protecting against,countering, or reversing the effects of aging.

SUMMARY

The present invention is based on the production and use of bloodproducts for treating and/or preventing age-related disorders, such ascognitive impairment conditions, age-related dementia, andneurodegenerative disease. The present invention recognizes, among otherthings, the need for new therapies for the treatment and/or preventionof cognitive impairment, age-related dementia, and neurodegenerativedisease. Derived from blood and blood plasma, the present compositionsof the invention relate to a solution for the failures and shortcomingsof current therapies through utilization of blood plasma fractionsexhibiting efficacy in the treatment and/or prevention of cognitiveimpairment, age-related dementia, and neurodegenerative disease.Additionally, the current invention relates to proteins identified inblood plasma fractions which either may exhibit efficacy as treatmentsor preventative agents for cognitive impairment and age-related dementiathemselves, or are targets for inhibition by additional agents.

The current invention also recognizes that differences in proteincontent between different blood plasma fractions (e.g. fractions,effluents, “Plasma Fractions,” Plasma Protein Fraction, Human AlbuminSolution) can be responsible for preventing and/or improving certaincognitive impairments and alleviating neurodegenerative disease. By wayof example, and not limitation, embodiments of the current inventiondemonstrate that mere higher albumin concentration of Human AlbuminSolution (HAS) preparations is not the driving force behind improvedcognition associated with Plasma Protein Fraction (PPF) preparationswith lower albumin concentrations.

Blood and blood plasma from young donors have exhibited improvement andreversal of the pre-existing effects of brain aging, including at themolecular, structural, functional, and cognitive levels. (Saul A.Villeda, et al. Young blood reverses age-related impairments incognitive function and synaptic plasticity in mice. Nature Medicine 20659-663 (2014)). The present invention relates to fractions andeffluents of the blood plasma, some of which have been traditionallyused to treat patient shock, and the discovery that they are effectiveas methods of treatment of aging-associated cognitive impairment.

In accordance with aspects of the invention, then, methods of treatmentof aging-associated cognitive impairment, age-related dementia, and/orneurodegenerative disease using blood product fractions of blood plasmaare provided. Aspects of the methods include administering a bloodplasma fraction to an individual suffering from or at risk of developingaging-associated cognitive impairment or neurodegenerative disease.Additional aspects of the methods include administering a blood plasmafraction derived from a pool of donors of a specific age range to anindividual suffering from or at risk of developing aging-associatedcognitive impairment. Also provided are reagents, devices, and kitsthereof that find use in practicing the subject methods.

In an embodiment, the blood plasma fraction may be, for example, one ofseveral blood plasma fractions obtained from a blood fractionationprocess, such as the Cohn fractionation process described below. Inanother embodiment, the blood plasma fraction may be of the type, hereinreferred to as “Plasma Fraction,” which is a solution comprised ofnormal human albumin, alpha and beta globulins, gamma globulin, andother proteins, either individually or as complexes. In anotherembodiment, the blood plasma fraction may be a type of blood plasmafraction known to those having skill in the art as a “Plasma ProteinFraction” (PPF). In another embodiment, the blood plasma fraction may bea “Human Albumin Solution” (HAS) fraction. In yet another embodiment,the blood plasma fraction may one in which substantially all of theclotting factors are removed in order to retain the efficacy of thefraction with reduced risk of thromboses. Embodiments of the inventionmay also include administering, for example, a fraction derived from ayoung donor or pools of young donors. Another embodiment of theinvention may include the monitoring of cognitive improvement in asubject treated with a blood plasma fraction.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate embodiments of the invention and,together with the description, serve to explain the invention. Thesedrawings are offered by way of illustration and not by way oflimitation: it is emphasized that the various features of the drawingsmay not be to-scale.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. FIG. 1 shows the time spent rearing by control, PPPF1, orHAS1-treated 3-month or 13-month-old NSG mice that were placed in anOpen Field chamber for 15 minutes.

FIG. 2. FIG. 2 shows the movement velocity of control, PPPF1, orHAS1-treated 3-month or 13-month-old NSG mice that were placed in anOpen Field chamber for 15 minutes.

FIG. 3. FIG. 3 shows the movement distance traveled of control, PPPF1,or HAS1-treated 3-month or 13-month-old NSG mice that were placed in anOpen Field chamber for 15 minutes.

FIG. 4. FIG. 4 shows the time spent in the novel arm by 3-month or13-month-old NSG mice in the cued Y-maze test that were treated withcontrol, PPPF1, or HAS1.

FIG. 5. FIG. 5 shows the ratio of time spent by 3-month or 13-month-oldNSG mice in the novel versus familiar arms (ratio of novel:familiar) ofthe cued Y-maze test, the mice having been treated with control, PPPF1,or HAS1.

FIG. 6. FIG. 6 shows the movement velocity of control, PPPF1, orHAS1-treated 3-month or 13-month-old NSG mice in the cued Y-maze test.

FIG. 7. FIG. 7 shows the movement distance traveled of 3-month or13-month-old NSG mice in the cued Y-maze test, the mice having beentreated with control, PPPF1, or HAS1.

FIG. 8A. FIG. 8A shows the per cent of time freezing in the contextualfear conditioning test for memory by 3-month and 13-month-old NSG micetreated with control, PPF1, or HAS1.

FIG. 8B. FIG. 8B shows the per cent of time freezing in the auditorycued fear conditioning test for memory by 3-month and 13-month-old NSGmice treated with control, PPF1, or HAS1.

FIG. 9. FIG. 9 quantifies per cent of time freezing during the last 90seconds of the cued fear conditioning test for memory in 3-month and13-month-old NSG mice treated with control, PPPF1, or HAS1.

FIG. 10A. FIG. 10A charts the Barnes maze latency which tests forspatial memory. The latency to reach the target hole in 3-month and13-month-old NSG mice treated with control, PPPF1, or HAS1 is reported.

FIG. 10B. FIG. 10B quantifies the average of the last 3 trials depictedin FIG. 10A.

FIG. 11A. FIG. 11A quantifies the number of cells positively stainingfor Doublecortin (Dcx), a marker for newborn neurons in the dentategyrus of 3-month and 13-month-old NSG mice treated with control, PPPF1,or HAS1 twice per week for up to 6 months.

FIG. 11B. FIG. 11B quantifies the number of cells positively stainingfor Ki67, a marker for proliferating cells in the dentate gyms of3-month and 13-month-old NSG mice treated with control, PPPF1, or HAS1twice per week for up to 6 months.

FIG. 12. FIG. 12 quantifies the number of cells positively staining forDcx in 13-month-old NSG mice treated with control, PPPF1, 1×concentrated HAS1, or 5× concentrated HAS1 three times per week for fiveweeks.

FIG. 13. FIG. 13 quantifies the number of cells positively staining forKi67 in 13-month-old NSG mice treated with control, PPPF1, 1×concentrated HAS1, or 5× concentrated HAS1 three times per week for fiveweeks.

FIG. 14A. FIG. 14A shows the number of rearing bouts in an Open Fieldchamber in NODscid mice treated twice weekly via intravenous tail veininjection with either saline (control) or PPF1 starting at 6 months ofage. Rearing was measured for a span of 15 minutes once mice were placedin the Open Field chamber.

FIG. 14B. FIG. 14B reports the movement velocity in an Open Fieldchamber of mice treated twice weekly via intravenous tail vein injectionwith either saline (control) or PPF1 starting at 6 months of age.Velocity was measured for a span of 15 minutes once mice were placed inthe Open Field chamber.

FIG. 14C. FIG. 14C reports the distance traveled in an Open Fieldchamber of mice treated twice weekly via intravenous tail vein injectionwith either saline (control) or PPF1 starting at 6 months of age.Velocity was measured for a span of 15 minutes once mice were placed inthe Open Field chamber.

FIG. 15. FIG. 15 depicts the Barnes maze latency andhippocampal-dependent spatial learning and memory. The latency to reachthe target hole in aged NSG mice (aged 12 months) treated with 150 μLsaline control, young plasma, Effluent I, or Effluent II/III isreported.

FIG. 16. FIG. 16 reports the effect of young human plasma, PPPF1, andsaline control on hippocampal-dependent spatial learning and memory inmale aged NSG mice (aged 12 months). The mice were treated with 150 μLof clarified young human plasma (young plasma), PPPF1, or saline threetimes per week (i.v.) for 4 weeks, and then twice per week during weeks5 and 6, which were the weeks in which testing was performed isreported. The latency to reach the Barnes Maze hold for each treatmentgroup is reported.

FIG. 17. FIG. 17 reports the effect of young human plasma, PPPF1, andsaline control on the average latency to find the Barnes Maze targethole for the last three trials for each day of testing. Aged NSG mice(aged 12 months) were treated with 150 μL of clarified young humanplasma (young plasma), PPPF1, or saline three times per week (i.v.) for4 weeks, and were subsequently treated twice per week during weeks 5 and6, which were the weeks in which testing was performed.

FIG. 18. FIG. 18 reports the effect of young human plasma, PPPF1, andsaline control on cell survival as determined by BrdU detection. AgedNSG mice (aged 12 months) were treated with 150 μL of clarified younghuman plasma (young plasma), PPPF1, or saline three times per week(i.v.) for 4 weeks, and were subsequently treated twice per week duringweeks 5 and 6, which were the weeks in which behavioral testing wasperformed. Hippocampal sections were analyzed after sacrifice.

FIG. 19. FIG. 19 shows the effects of control, PPPF1, and HAS1 onneurosphere proliferation in cortex culture. The figure shows exampleimages of neurospheres from cortical cultures after 21 days in vitro,imaged for Tuj1, DAPI, or Tuj1 and DAPI.

FIG. 20. FIG. 20 shows the effects of control, PPPF1, and HAS1 on netneurite length in cortex culture.

FIG. 21. FIG. 21 shows effects of vehicle, PPPF1, and HAS1 on sphere andprocess growth in cortex culture. Yellow shading highlights spheres, andpink shading highlights neurites determined by an IncuCyte softwarealgorithm (Essen BioScience, Inc., Ann Arbor, Mich.).

FIG. 22A. FIG. 22A shows the quantification of neurosphere number aspercent of vehicle from cortices from E14-15 mouse embryos suspended inneural basal media supplemented with B27 and 2 mM Glutamax (vehicle),PPF1 (10% of a 5% stock solution), or HAS1 (10% of a 5% stock solution).

FIG. 22B. FIG. 21B shows the quantification of neurite length as percentof vehicle from cortices from E14-15 mouse embryos suspended in neuralbasal media supplemented with B27 and 2 mM Glutamax (vehicle), PPF1 (10%of a 5% stock solution), or HAS1 (10% of a 5% stock solution).

FIG. 22C. FIG. 21C shows the quantification of neurite branch points aspercent of vehicle from cortices from E14-15 mouse embryos suspended inneural basal media supplemented with B27 and 2 mM Glutamax (vehicle),PPF1 (10% of a 5% stock solution), or HAS1 (10% of a 5% stock solution).

FIG. 22D. FIG. 22D shows the quantification of neurosphere size aspercent of vehicle from cortices from E14-15 mouse embryos suspended inneural basal media supplemented with B27 and 2 mM Glutamax (vehicle),PPF1 (10% of a 5% stock solution), or HAS1 (10% of a 5% stock solution).

FIG. 23. FIG. 23 shows the quantification of the number of neurospheresstaining positive for Sox2, which were treated with control vehicle(neural basal media supplemented with B27 and 2 mM Glutamax), PPF1 (10%of a 5% stock solution), or HAS1 (10% of a 5% stock solution). Sox2staining is an indicator of a neurosphere's potential for neurogenesis.

DETAILED DESCRIPTION 1. Introduction

The present invention relates to the identification and discovery ofmethods and compositions for the treatment and/or prevention ofcognitive impairment, including age-associated dementia andneurodegenerative disease. Described herein are methods and compositionsfor the treatment of subjects suffering from such disorders, which areaspects of the present invention. The methods and compositions describedherein are useful in: preventing cognitive impairment, age-associateddementia, and neurodegenerative disease; ameliorating the symptoms ofcognitive impairment, age-associated dementia, and neurodegenerativedisease; slowing progression of aging-associated cognitive impairment,age-associated dementia, and neurodegenerative disease; and/or reversingthe progression of aging-associated cognitive impairment, age-associateddementia, and neurodegenerative disease. An implementation of theinvention includes using blood plasma fractions as treatment, such asone or more fractions or effluents obtained from blood fractionationprocesses, e.g., like the Cohn fractionation process described below. Anembodiment of the invention includes using Plasma Fraction (a solutioncomprised of normal human albumin, alpha and beta globulins, gammaglobulin, and other proteins either individually or as complexes,hereinafter referred to as “Plasma Fraction”). Another embodiment of theinvention includes using Plasma Protein Fraction (PPF) as treatment.Another embodiment of the invention includes using Human AlbuminSolution (HAS) fraction as treatment. Yet another embodiment includesusing effluents from blood fractionation processes such as Effluent I orEffluent II/III described below. An additional embodiment includes ablood plasma fraction from which substantially all the clotting factorshave been removed in order to retain efficacy while reducing the risk ofthromboses (for example, see U.S. Patent Application No. 62/236,710,which is incorporated by reference in its entirety herein).

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is alsounderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein have discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or the spirit of thepresent invention. Any recited method can be carried out in the order ofevents recited or in any other order which is logically possible.

2. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art to which the invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, some potentialand preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those having skill in the art, and so forth.

In describing methods of the present invention, the terms “host”,“subject”, “individual” and “patient” are used interchangeably and referto any mammal in need of such treatment according to the disclosedmethods. Such mammals include, e.g., humans, ovines, bovines, equines,porcines, canines, felines, non-human primate, mice, and rats. Incertain embodiments, the subject is a non-human mammal. In someembodiments, the subject is a farm animal. In other embodiments, thesubject is a pet. In some embodiments, the subject is mammalian. Incertain instances, the subject is human. Other subjects can includedomestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats,horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g.,as in animal models of disease), as well as non-human primates (e.g.,chimpanzees, and monkeys). As such, subjects of the invention, includebut are not limited to mammals, e.g., humans and other primates, such aschimpanzees and other apes and monkey species; and the like, where incertain embodiments the subject are humans. The term subject is alsomeant to include a person or organism of any age, weight or otherphysical characteristic, where the subjects may be an adult, a child, aninfant or a newborn.

By a “young” or “young individual” it is meant an individual that is ofchronological age of 40 years old or younger, e.g., 35 years old oryounger, including 30 years old or younger, e.g., 25 years old oryounger or 22 years old or younger. In some instances, the individualthat serves as the source of the young plasma-comprising blood productis one that is 10 years old or younger, e.g., 5 years old or younger,including 1-year-old or younger. In some instances, the subject is anewborn and the source of the plasma product is the umbilical cord,where the plasma product is harvested from the umbilical cord of thenewborn. As such, “young” and “young individual” may refer to a subjectthat is between the ages of 0 and 40, e.g., 0, 1, 5, 10, 15, 20, 25, 30,35, or 40 years old. In other instances, “young” and “young individual”may refer to a biological (as opposed to chronological) age such as anindividual who has not exhibited the levels of inflammatory cytokines inthe plasma exhibited in comparatively older individuals. Conversely,these “young” and “young individual” may refer to a biological (asopposed to chronological) age such as an individual who exhibits greaterlevels of anti-inflammatory cytokines in the plasma compared to levelsin comparatively older individuals. By way of example, and notlimitation, the inflammatory cytokine is Eotaxin, and the folddifference between a young subject or young individual and olderindividuals is at least 20%. Similarly, the fold difference betweenolder and younger individuals in other inflammatory cytokines may beused to refer to a biological age. (See U.S. patent application Ser. No.13/575,437 which is herein incorporated by reference). Usually, theindividual is healthy, e.g., the individual has no hematologicalmalignancy or autoimmune disease at the time of harvest.

By “an individual suffering from or at risk of suffering from anaging-associated cognitive impairment” is meant an individual that isabout more than 50% through its expected lifespan, such as more than60%, e.g., more than 70%, such as more than 75%, 80%, 85%, 90%, 95% oreven 99% through its expected lifespan. The age of the individual willdepend on the species in question. Thus, this percentage is based on thepredicted life-expectancy of the species in question. For example, inhumans, such an individual is 50 year old or older, e.g., 60 years oldor older, 70 years old or older, 80 years old or older, 90 years old orolder, and usually no older than 100 years old, such as 90 years old.,i.e., between the ages of about 50 and 100, e.g., 50 . . . 55 . . . 60 .. . 65 . . . 70 . . . 75 . . . 80 . . . 85 . . . 90 . . . 95 . . . 100years old or older, or any age between 50-1000, that suffers from anaging-associated condition as further described below, e.g., cognitiveimpairment associated with the natural aging process; an individual thatis about 50 years old or older, e.g., 60 years old or older, 70 yearsold or older, 80 years old or older, 90 years old or older, and usuallyno older than 100 years old, i.e., between the ages of about 50 and 100,e.g., 50 . . . 55 . . . 60 . . . 65 . . . 70 . . . 75 . . . 80 . . . 85. . . 90 . . . 95 . . . 100 years old, that has not yet begun to showsymptoms of an aging-associated condition e.g., cognitive impairment; anindividual of any age that is suffering from a cognitive impairment dueto an aging-associated disease, as described further below, and anindividual of any age that has been diagnosed with an aging-associateddisease that is typically accompanied by cognitive impairment, where theindividual has not yet begun to show symptoms of cognitive impairment.The corresponding ages for non-human subjects are known and are intendedto apply herein.

As used herein, “treatment” refers to any of (i) the prevention of thedisease or disorder, or (ii) the reduction or elimination of symptoms ofthe disease or disorder. Treatment may be effected prophylactically(prior to the onset of disease) or therapeutically (following the onsetof the disease). The effect may be prophylactic in terms of completelyor partially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. Thus, the term “treatment”as used herein covers any treatment of an aging-related disease ordisorder in a mammal, and includes: (a) preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development; or (c) relieving the disease, i.e., causingregression of the disease. Treatment may result in a variety ofdifferent physical manifestations, e.g., modulation in gene expression,rejuvenation of tissue or organs, etc. The therapeutic agent may beadministered before, during or after the onset of disease. The treatmentof ongoing disease, where the treatment stabilizes or reduces theundesirable clinical symptoms of the patient, is of particular interest.Such treatment may be performed prior to complete loss of function inthe affected tissues. The subject therapy may be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

In some embodiments, the aging-associated condition that is treated isan aging-associated impairment in cognitive ability in an individual. Bycognitive ability, or “cognition,” it is meant the mental processes thatinclude attention and concentration, learning complex tasks andconcepts, memory (acquiring, retaining, and retrieving new informationin the short and/or long term), information processing (dealing withinformation gathered by the five senses), visuospatial function (visualperception, depth perception, using mental imagery, copying drawings,constructing objects or shapes), producing and understanding language,verbal fluency (word-finding), solving problems, making decisions, andexecutive functions (planning and prioritizing). By “cognitive decline”,it is meant a progressive decrease in one or more of these abilities,e.g., a decline in memory, language, thinking, judgment, etc. By “animpairment in cognitive ability” and “cognitive impairment”, it is meanta reduction in cognitive ability relative to a healthy individual, e.g.,an age-matched healthy individual, or relative to the ability of theindividual at an earlier point in time, e.g., 2 weeks, 1 month, 2months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years ormore previously. By “aging-associated cognitive impairment,” it is meantan impairment in cognitive ability that is typically associated withaging, including, for example, cognitive impairment associated with thenatural aging process, e.g., mild cognitive impairment (M.C.I.); andcognitive impairment associated with an aging-associated disorder, thatis, a disorder that is seen with increasing frequency with increasingsenescence, e.g., a neurodegenerative condition such as Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, Huntingtondisease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,myotonic dystrophy, vascular dementia, and the like.

Blood Products Comprising Plasma Components. In practicing the subjectmethods, a blood product comprising plasma components is administered toan individual in need thereof, e.g., an individual suffering or at riskof suffering from a cognitive impairment and/or age-related dementia. Assuch, methods according to embodiments of the invention includingadministering a blood product comprising plasma components from anindividual (the “donor individual”, or “donor”) to an individual atleast at risk of suffering or suffering from cognitive impairment and/orage-related dementia (the “recipient individual” or “recipient”). By a“blood product comprising plasma components,” it is meant any productderived from blood that comprises plasma (e.g. whole blood, bloodplasma, or fractions thereof). The term “plasma” is used in itsconventional sense to refer to the straw-colored/pale-yellow liquidcomponent of blood composed of about 92% water, 7% proteins such asalbumin, gamma globulin, anti-hemophilic factor, and other clottingfactors, and 1% mineral salts, sugars, fats, hormones and vitamins.Non-limiting examples of plasma-comprising blood products suitable foruse in the subject methods include whole blood treated withanti-coagulant (e.g., EDTA, citrate, oxalate, heparin, etc.), bloodproducts produced by filtering whole blood to remove white blood cells(“leukoreduction”), blood products consisting ofplasmapheretically-derived or apheretically-derived plasma, fresh-frozenplasma, blood products consisting essentially of purified plasma, andblood products consisting essentially of plasma fractions. In someinstances, plasma product that is employed is a non-whole blood plasmaproduct, by which is meant that the product is not whole blood, suchthat it lacks one or more components found in whole blood, such aserythrocytes, leukocytes, etc., at least to the extent that thesecomponents are present in whole blood. In some instances, the plasmaproduct is substantially, if not completely, acellular, where in suchinstances the cellular content may be 5% by volume or less, such as 1%or less, including 0.5% or less, where in some instances acellularplasma fractions are those compositions that completely lack cells,i.e., they include no cells.

Collection of blood products comprising plasma components. Embodimentsof the methods described herein include administration of blood productscomprising plasma components which can be derived from donors, includinghuman volunteers. The term, “human-derived” can refer to such products.Methods of collection of plasma comprising blood products from donorsare well-known in the art. (See, e.g., AABB TECHNICAL MANUAL, (Mark A.Fung, et al., eds., 18th ed. 2014), herein incorporated by reference).

In one embodiment, donations are obtained by venipuncture. In anotherembodiment, the venipuncture is only a single venipuncture. In anotherembodiment, no saline volume replacement is employed. In an embodiment,the process of plasmapheresis is used to obtain the plasma comprisingblood products. Plasmapheresis can comprise the removal of aweight-adjusted volume of plasma with the return of cellular componentsto the donor. In the embodiment, sodium citrate is used duringplasmapheresis in order to prevent cell clotting. The volume of plasmacollected from a donor is preferably between 690 to 880 mL after citrateadministration, and preferably coordinates with the donor's weight.

3. Blood Plasma Fractions

During the Second World War, there arose a need for a stable plasmaexpander which could be employed in the battlefield when soldiers lostlarge amounts of blood. As a result, methods of preparing freeze-driedplasma were developed. However, use of freeze-dried plasma was difficultin combat situations since reconstitution required sterile water. As analternative, Dr. E. J. Cohn suggested that albumin could be used, andprepared a ready-to-use stable solution that could be introducedimmediately for treatment of shock. (See JOHAN VANDERSANDE, CURRENTAPPROACHES TO THE PREPARATION OF PLASMA FRACTIONS in (BIOTECHNOLOGY OFBLOOD) 165 (Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's procedure ofpurifying plasma fractions utilized cold ethanol for its denaturingeffect, and employs changes in pH and temperature to achieve separation.

An embodiment of the methods described herein includes theadministration of plasma fractions to a subject. Fractionation is theprocess by which certain protein subsets are separated from plasma.Fractionation technology is known in the art and relies on stepsdeveloped by Cohn et al. during the 1940s. (E. Cohn, Preparation andproperties of serum and plasma proteins. IV. A system for the separationinto fractions of the protein and lipoprotein components of biologicaltissues and fluids. 68 J Am Chem Soc 459 (1946), herein incorporated byreference). Several steps are involved in this process, each stepinvolving specific ethanol concentrations as well as pH, temperature,and osmolality shifts which result in selective protein precipitation.Precipitates are also separated via centrifugation or precipitation. Theoriginal “Cohn fractionation process” involved separation of proteinsthrough precipitates into five fractions, designated fraction I,fraction II+III, fraction IV-1, fraction IV-4 and fraction V. Albuminwas the originally identified endpoint (fraction V) product of thisprocess. In accordance with embodiments of the invention, each fraction(or effluent from a prior separation step) contains or potentiallycontains therapeutically-useful protein fractions. (See Thierry Burma,Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101(2007); Adil Denizli, Plasma fractionation: conventional andchromatographic methods for albumin purification, 4 J. Biol. & Chem.315, (2011); and T. Brodniewicz-Proba, Human Plasma Fractionation andthe Impact of New Technologies on the Use and Quality of Plasma-derivedProducts, 5 Blood Reviews 245 (1991), and U.S. Pat. Nos. 3,869,431,5,110,907, 5,219,995, 7,531,513, and 8,772,461 which are hereinincorporated by reference). Adjustment of the above experimentalparameters can be made in order to obtain specific protein fractions.

More recently, fractionation has reached further complexity, and assuch, comprises additional embodiments of the invention. This recentincrease in complexity has occurred through: the introduction ofchromatography resulting in isolation of new proteins from existingfractions like cryoprecipitate, cryo-poor plasma, and Cohn fractions;increasing IgG recovery by integrating chromatography and the ethanolfractionation process; and viral reduction/inactivation/removal. (Id.)In order to capture proteins at physiological pH and ionic strength,anion-exchange chromatography can be utilized. This preserves functionalactivity of proteins and/or protein fractions. Heparin and monoclonalantibodies are also used in affinity chromatography. One of ordinaryskill in the art would recognize that the parameters described above maybe adjusted to obtain specifically-desired plasma protein-containingfractions.

In an embodiment of the invention, blood plasma is fractionated in anindustrial setting. Frozen plasma is thawed at 1° C. to 4° C. Continuousrefrigerated centrifugation is applied to the thawed plasma andcryoprecipitate isolated. Recovered cryoprecipitate is frozen at −30° C.or lower and stored. The cryoprecipitate-poor (“cryo-poor”) plasma isimmediately processed for capture (via, for example, primarychromatography) of labile coagulation factors such as factor IX complexand its components as well as protease inhibitors such as antithrombinand C1 esterase inhibitor. Serial centrifugation and precipitateisolation can be applied in subsequent steps. Such techniques are knownto one of ordinary skill in the art and are described, for example, inU.S. Pat. Nos. 4,624,780, 5,219,995, 5,288,853, and U.S. patentapplication nos. 20140343255 and 20150343025, which disclosures areincorporated by reference in their entirety herein.

In an embodiment of the invention, the plasma fraction may comprise aplasma fraction containing a substantial concentration of albumin. Inanother embodiment of the invention, the plasma fraction may comprise aplasma fraction containing a substantial concentration of IgG orintravenous immune globulin (IGIV) (e.g. Gamunex-C®). In anotherembodiment of the invention the plasma fraction may comprise an IGIVplasma fraction, such as Gamunex-C® which has been substantiallydepleted of immune globulin (IgG) by methods well-known by one ofordinary skill in the art, such as for example, Protein A-mediateddepletion. (See Keshishian, H., et al., Multiplexed, QuantitativeWorkflow for Sensitive Biomarker Discovery in Plasma Yields NovelCandidates for Early Myocardial Injury, Molecular & Cellular Proteomics,14 at 2375-93 (2015)). In an additional embodiment, the blood plasmafraction may be one in which substantially all the clotting factors areremoved in order to retain the efficacy of the fraction with reducedrisk of thromboses. For example, the plasma fraction may be a plasmafraction as described in U.S. Patent No. 62/376,529 filed on Aug. 18,2016; the disclosure of which is incorporated by reference in itsentirety herein.

4. Albumin Products

To those having ordinary skill in the art, there are two generalcategories of Albumin Plasma Products (“APP”): plasma protein fraction(PPF) and human albumin solution (HAS). PPF is derived from a processwith a higher yield than HAS, but has a lower minimum albumin puritythan HAS (>83% for PPF and >95% for HAS). (Production of human albuminsolution: a continually developing colloid, P. Matejtschuk et al.,British J. of Anaesthesia 85(6): 887-95, at 888 (2000)). Additionally,some have noted that PPF has a disadvantage because of the presence ofprotein “contaminants” such as PKA. Id. As a consequence, PPFpreparations have lost popularity as Albumin Plasma Products, and haveeven been delisted from certain countries' Pharmacopoeias. Id. Contraryto these concerns, the invention makes beneficial use of these“contaminants.” Besides α, β, and γ globulins, as well as theaforementioned PKA, the methods of the invention utilize additionalproteins or other factors within the “contaminants” that promoteprocesses such as neurogenesis, neuronal cell survival, and improvedcognition.

Those of skill in the art will recognize that there are, or have been,several commercial sources of PPF (the “Commercial PPF Preparations.”)These include Plasma-Plex™ PPF (Armour Pharmaceutical Co., Tarrytown,N.Y.), Plasmanate™ PPF (Grifols, Clayton, N.C.), Plasmatein™ (AlphaTherapeutics, Los Angeles, Calif.), and ProtenateTM PPF (Baxter Labs,Inc. Deerfield, Ill.).

Those of skill in the art will also recognize that there are, or havebeen, several commercial sources of HAS (the “Commercial HASPreparations.”) These include AlbuminarTM (CSL Behring), AlbuRx™ (CSLBehring), Albutein™ (Grifols, Clayton, N.C.), Buminate™ (Baxatla, Inc.,Bannockburn, Ill.), Flexbumin™ (Baxatla, Inc., Bannockburn, Ill.), andPlasbumin™ (Grifols, Clayton, N.C.).

A. Plasma Protein Fraction (Human) (PPF)

According to the United States Food and Drug Administration (“FDA”),“Plasma Protein Fraction (Human),” or PPF, is the proper name of theproduct defined as “a sterile solution of protein composed of albuminand globulin, derived from human plasma.” (Code of Federal Regulations“CFR” 21 CFR 640.90 which is herein incorporated by reference). PPF'ssource material is plasma recovered from Whole Blood prepared asprescribed in 21 CFR 640.1-640.5 (incorporated by reference herein), orSource Plasma prepared as prescribed in 21 CFR 640.60-640.76(incorporated by reference herein).

PPF is tested to determine it meets the following standards as per 21CFR 640.92 (incorporated by reference herein):

-   -   (a) The final product shall be a 5.0+/−0.30 percent solution of        protein; and    -   (b) The total protein in the final product shall consist of at        least 83 percent albumin, and no more than 17 percent globulins.        No more than 1 percent of the total protein shall be gamma        globulin. The protein composition is determined by a method that        has been approved for each manufacturer by the Director, Center        for Biologics Evaluation and Research, Food and Drug        Administration.

As used herein, “Plasma Protein Fraction” or “PPF” refers to a sterilesolution of protein composed of albumin and globulin, derived from humanplasma, with an albumin content of at least 83% with no more than 17%globulins (including α1, α2, β, and γ globulins) and other plasmaproteins, and no more than 1% gamma globulin as determined byelectrophoresis. (Hink, J. H., Jr., et al., Preparation and Propertiesof a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174)(1957)). PPF can also refer to a solid form, which when suspended insolvent, has similar composition. The total globulin fraction can bedetermined through subtracting the albumin from the total protein.(Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY,PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, Walker H K, Hall W D,Hurst J D, eds. (1990)).

B. Albumin (Human) (HAS)

According to the FDA, “Albumin (Human)” (also referred to herein as“HAS”) is the proper name of the product defined as “sterile solution ofthe albumin derived from human plasma.” (Code of Federal Regulations“CFR” 21 CFR 640.80 which is herein incorporated by reference.) Thesource material for Albumin (Human) is plasma recovered from Whole Bloodprepared as prescribed in 21 CFR 640.1-640.5 (incorporated by referenceherein), or Source Plasma prepared as prescribed in 21 CFR 640.60-640.76(incorporated by reference herein). Other requirements for Albumin(Human) are listed in 21 CFR 640.80-640.84 (incorporated by referenceherein).

Albumin (Human) is tested to determine if it meets the followingstandards as per 21 CFR 640.82:

(a) Protein concentration. Final product shall conform to one of thefollowing concentrations: 4.0+/−0.25 percent; 5.0+/−0.30 percent;20.0+/−1.2 percent; and 25.0+/−1.5 percent solution of protein.

(b) Protein composition. At least 96 percent of the total protein in thefinal product shall be albumin, as determined by a method that has beenapproved for each manufacturer by the Director, Center for BiologicsEvaluation and Research, Food and Drug Administration.

As used herein, “Albumin (Human)” or “HAS” refers to a to a sterilesolution of protein composed of albumin and globulin, derived from humanplasma, with an albumin content of at least 95%, with no more than 5%globulins (including α1, α2, β, and γ globulins) and other plasmaproteins. HAS can also refer to a solid form, which when suspended insolvent, has similar composition. The total globulin fraction can bedetermined through subtracting the albumin from the total protein.

As can be recognized by one having ordinary skill in the art, PPF andHAS fractions can also be freeze-dried or in other solid form. Suchpreparations, with appropriate additives, can be used to make tablets,powders, granules, or capsules, for example. The solid form can beformulated into preparations for injection by dissolving, suspending oremulsifying them in an aqueous or non-aqueous solvent, such as vegetableor other similar oils, synthetic aliphatic acid glycerides, esters ofhigher aliphatic acids or propylene glycol; and if desired, withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

5. Clotting Factor-Reduced Fractions

Another embodiment of the invention uses a blood plasma fraction fromwhich substantially all of the clotting factors are removed in order toretain the efficacy of the fraction with reduced risk of thromboses.Conveniently, the blood product can be derived from a young donor orpool of young donors, and can be rendered devoid of IgM in order toprovide a young blood product that is ABO compatible. Currently, plasmathat is transfused is matched for ABO blood type, as the presence ofnaturally occurring antibodies to the A and B antigens can result intransfusion reactions. IgM appears to be responsible for transfusionreactions when patients are given plasma that is not ABO matched.Removal of IgM from blood products or fractions helps eliminatetransfusion reactions in subjects who are administered the bloodproducts and blood plasma fractions of the invention.

Accordingly, in one embodiment, the invention is directed to a method oftreating or preventing an aging-related condition such as cognitiveimpairment or neurodegeneration in a subject. The method comprises:administering to the subject a blood product or blood fraction derivedfrom whole-blood from an individual or pool of individuals, wherein theblood product or blood fraction is substantially devoid of (a) at leastone clotting factor and/or (b) IgM. In some embodiments, theindividual(s) from whom the blood product or blood fraction is derivedare young individuals. In some embodiments, the blood product issubstantially devoid of at least one clotting factor and IgM. In certainembodiments, the blood product is substantially devoid of fibrinogen(Factor I). In additional embodiments, the blood product substantiallylacks erythrocytes and/or leukocytes. In further embodiments, the bloodproduct is substantially acellular. In other embodiments, the bloodproduct is derived from plasma. Such embodiments of the invention arefurther supported by U.S. Patent Application No. 62/376,529 filed onAug. 18, 2016, which is incorporated by reference in its entiretyherein.

6. Protein-Enriched Plasma Protein Products

Additional embodiments of the invention use plasma fractions withreduced albumin concentration compared to PPF, but with increasedamounts of globulins and other plasma proteins (what have been referredto by some as “contaminants”). The embodiments, as with PPF, HAS,Effluent I, and Effluent II/III are all effectively devoid of clottingfactors. Such plasma fractions are hereinafter referred to as“protein-enriched plasma protein products”. For example, an embodimentof the invention may use a protein-enriched plasma protein productcomprised of 82% albumin and 18% α, β, and γ globulins and other plasmaproteins. Another embodiment of the invention may use a protein-enrichedplasma protein product comprised of 81% albumin and 19% of α, β, and γglobulins and/or other plasma proteins. Another embodiment of theinvention may use a protein-enriched plasma protein product comprised of80% albumin and 20% of α, β, and γ globulins and/or other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 70-79% albumin anda corresponding 21-30% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 60-69% albumin anda corresponding 31-40% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 50-59% albumin anda corresponding 41-50% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 40-49% albumin anda corresponding 51-60% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 30-39% albumin anda corresponding 61-70% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 20-29% albumin anda corresponding 71-80% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 10-19% albumin anda corresponding 81-90% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 1-9% albumin and acorresponding 91-99% of α, β, and γ globulins and other plasma proteins.A further embodiment of the invention may use protein-enriched plasmaprotein products comprised of 0-1% albumin and 99-100% of α, β, and γglobulins and other plasma proteins

Embodiments of the invention described above may also have total gammaglobulin concentrations of 0-5%.

The specific concentrations of proteins in a plasma fraction may bedetermined using techniques well-known to a person having ordinary skillin the relevant art. By way of example, and not limitation, suchtechniques include electrophoresis, mass spectrometry, ELISA analysis,and Western blot analysis.

7. Preparation of Blood Plasma Fractions

Methods of preparing PPF and other plasma fractions are well-known tothose having ordinary skill in the art. An embodiment of the inventionallows for blood used in the preparation of human plasma proteinfraction to be collected in flasks with citrate or anticoagulant citratedextrose solution for inhibition of coagulation, with further separationof Fractions I, II+III, IV, and PPF as per the method disclosed in Hinket al. (See Hink, J. H., Jr., et al., Preparation and Properties of aHeat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957),herein incorporated by reference.) According to this method, the mixturecan be collected to 2-8° C. The plasma can then subsequently beseparated by centrifugation at 7° C., removed, and stored at −20° C. Theplasma can then be thawed at 37° C. and fractionated, preferably withineight hours after removal from −20° C. storage.

Plasma can be separated from Fraction I using 8% ethanol at pH 7.2 and atemperature at −2 to −2.5° C. with protein concentration of 5.1 to 5.6percent. Cold 53.3 percent ethanol (176 mL/L of plasma) with acetatebuffer (200 mL 4M sodium acetate, 230 mL glacial acetic acid quantumsatis to 1 L with H₂O) can be added using jets at a rate, for example,of 450 mL/minute during the lowering the plasma temperature to −2° C.Fraction I can be separated and removed from the effluent (Effluent I)through ultracentrifugation. Fibrinogen can be obtained from Fraction Ias per methods well-known to those having ordinary skill in the art.

Fraction II+III can be separated from Effluent I through adjustment ofthe effluent to 21 percent ethanol at pH 6.8, temperature at −6° C.,with protein concentration of 4.3 percent. Cold 95 percent ethanol (176mL/L of Effluent I) with 10 M acetic acid used for pH adjustment can beadded using jets at a rate, for example, of 500 mL/minute during thelowering of the temperature of Effluent I to −6° C. The resultingprecipitate (Fraction II+III) can be removed by centrifugation at −6° C.Gamma globulin can be obtained from Fraction II+III using methodswell-known to those having ordinary skill in the art.

Fraction IV-1 can be separated from Effluent II+III (“Effluent II/III”)through adjustment of the effluent to 19 percent ethanol at pH 5.2,temperature at −6° C., and protein concentration of 3 percent. H₂0 and10 M acetic acid used for pH adjustment can be added using jets whilemaintaining Effluent II/III at −6° C. for 6 hours. Precipitated FractionVI-1 can be settled at −6° C. for 6 hours and subsequently separatedfrom the effluent by centrifugation at the same temperature. Stableplasma protein fraction can be recovered from Effluent IV-1 throughadjustment of the ethanol concentration to 30 percent at pH 4.65,temperature −7° C. and protein concentration of 2.5 percent. This can beaccomplished by adjusting the pH of Effluent IV-1 with cold acid-alcohol(two parts 2 M acetic acid and one part 95 percent ethanol). Whilemaintaining a temperature of −7° C., to every liter of adjusted EffluentIV-1 170 mL cold ethanol (95%) is added. Proteins that precipitate canbe allowed to settle for 36 hours and subsequently removed bycentrifugation at −7° C.

The recovered proteins (stable plasma protein fraction) can be dried(e.g. by freeze drying) to remove alcohol and H₂0. The resulting driedpowder can be dissolved in sterile distilled water, for example using 15liters of water/kg of powder, with the solution adjusted to pH 7.0 with1 M NaOH. A final concentration of 5 per cent protein can be achieved byadding sterile distilled water containing sodium acetyl tryptophanate,sodium caprylate, and NaCl, adjusting to final concentrations of 0.004 Macetyl tryptophanate, 0.004 M caprylate, and 0.112 M sodium. Finally,the solution can be filtered at 10° C. to obtain a clear solution andsubsequently heat-treated for inactivation of pathogens at 60° C. for atleast 10 hours.

One having ordinary skill in the art would recognize that each of thedifferent fractions and effluents described above could be used with themethods of the invention to treat disease. For example, and not by wayof limitation, Effluents I or Effluent II/III may be utilized to treatsuch diseases as cognitive and neurodegenerative disorders and areembodiments of the invention.

The preceding methods of preparing blood plasma fractions and plasmaprotein fraction (PPF) are only exemplary and involves merelyembodiments of the invention. One having ordinary skill in the art wouldrecognize that these methods can vary. For example, pH, temperature, andethanol concentration, among other things can be adjusted to producedifferent variations of plasma fractions and plasma protein fraction inthe different embodiments and methods of the invention. In anotherexample, additional embodiments of the invention contemplate the use ofnanofiltration for the removal/inactivation of pathogens from plasmafractions and plasma protein fraction.

An additional embodiment of the invention contemplates methods andcomposition using and/or comprising additional blood plasma fractions.For example, the invention, among other things, demonstrates thatspecific concentrations of albumin are not critical for improvingcognitive activity. Hence, fractions with reduced albumin concentration,such as those fractions having below 83% albumin, are contemplated bythe invention.

8. Treatment

Aspects of the methods of the inventions described herein includetreatment of a subject with a plasma comprising blood product, such as ablood plasma fraction, e.g., as described above. An embodiment includestreatment of a human subject with a plasma comprising blood product. Oneof skill in the art would recognize that methods of treatment ofsubjects with plasma comprising blood products are recognized in theart. By way of example, and not limitation, one embodiment of themethods of the inventions described herein is comprised of administeringfresh frozen plasma to a subject for treatment and/or prevention ofcognitive impairment and/or age-related dementia. In one embodiment, theplasma comprising blood product is administered immediately, e.g.,within about 12-48 hours of collection from a donor, to the individualsuffering or at risk from a cognitive impairment and/or age-relateddementia. In such instances, the product may be stored underrefrigeration, e.g., 0-10° C. In another embodiment, fresh frozen plasmais one that has been stored frozen (cryopreserved) at −18° C. or colder.Prior to administration, the fresh frozen plasma is thawed and oncethawed, administered to a subject 60-75 minutes after the thawingprocess has begun. Each subject preferably receives a single unit offresh frozen plasma (200-250 mL), the fresh frozen plasma preferablyderived from donors of a pre-determined age range. In one embodiment ofthe invention, the fresh frozen plasma is donated by (derived from)young individuals. In another embodiment of the invention, the freshfrozen plasma is donated by (derived from) donors of the same gender. Inanother embodiment of the invention, the fresh frozen plasma is donatedby (derived from) donors of the age range between 18-22 years old. Inone embodiment, subjects are treated twice per week with 3-4 daysbetween infusions. In an embodiment of the invention, treatment persistsuntil a specific endpoint is reached.

In an embodiment of the invention, the plasma comprising blood productsare screened after donation by blood type. In another embodiment of theinvention, the plasma comprising blood products are screened forinfectious disease agents such as HIV I & II, HBV, HCV, HTLV I & II,anti-HBc per the requirements of 21 CFR 640.33 and recommendationscontained in FDA guidance documents.

In yet another embodiment of the invention, the subject is treated witha “Plasma Fraction.” In an embodiment of the invention, the PlasmaFraction is PPF or HAS. In a further embodiment of the invention, thePlasma Fraction is one of the Commercial PPF Preparations of theCommercial HAS Preparations. In another embodiment of the invention thePlasma Fraction is a PPF or HAS derived from a pool of individuals of aspecific age range, such as young individuals, or is a modified PPF orHAS fraction which has been subjected to additional fractionation orprocessing (e.g. PPF or HAS with one or more specific proteins partiallyor substantially removed). In another embodiment of the invention, thePlasma Fraction is an IGIV plasma fraction which has been substantiallydepleted of immune globulin (IgG). A blood fraction which is“substantially depleted” or which has specific proteins “substantiallyremoved,” such as IgG, refers to a blood fraction containing less thanabout 50% of the amount that occurs in the reference product or wholeblood plasma, such as less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integerbetween these values, as measured using standard assays well known inthe art.

9. Monitoring

Another aspect of the present invention relates to methods of monitoringthe effect of a medication on a subject for treating cognitiveimpairment and/or age-related dementia, the method comprising comparingcognitive function before and after treatment. Those having ordinaryskill in the art recognize that there are well-known methods ofevaluating cognitive function. For example, and not by way oflimitation, the method may comprise evaluation of cognitive functionbased on medical history, family history, physical and neurologicalexaminations by clinicians who specialize dementia and cognitivefunction, laboratory tests, and neuropsychological assessment.Additional embodiments which are contemplated by the invention include:the assessment of consciousness, such as using the Glasgow Coma Scale(EMV); mental status examination, including the abbreviated mental testscore (AMTS) or mini-mental state examination (MMSE) (Folstein et al.,J. Psychiatr. Res 1975; 12:1289-198); global assessment of higherfunctions; estimation of intracranial pressure such as by fundoscopy.

In one embodiment, examinations of peripheral nervous system may be usedto evaluate cognitive function, including any one of the followings:sense of smell, visual fields and acuity, eye movements and pupils(sympathetic and parasympathetic), sensory function of face, strength offacial and shoulder girdle muscles, hearing, taste, pharyngeal movementand reflex, tongue movements, which can be tested individually (e.g. thevisual acuity can be tested by a Snellen chart; a reflex hammer usedtesting reflexes including masseter, biceps and triceps tendon, kneetendon, ankle jerk and plantar (i.e. Babinski sign); Muscle strengthoften on the MRC scale 1 to 5; Muscle tone and signs of rigidity.

10. Administration

In practicing methods of the invention, a blood plasma fraction isadministered to the subject. In an embodiment, the blood plasma fractionis administered by intravenous infusion. The rate of infusion may vary,but in one embodiment of the invention, the infusion rate is 5-8mL/minute. Those having ordinary skill in the art will recognize thatthe infusion rate can depend upon the subject's condition and responseto administration.

In those embodiments where an effective amount of an active agent isadministered to the adult mammal, the amount or dosage is effective whenadministered for a suitable period of time, such as one week or longer,including two weeks or longer, such as 3 weeks or longer, one month orlonger, 2 months or longer, 3 months or longer, 4 months or longer, 5months or longer, 6 months or longer, 1 year or longer etc., so as toevidence a reduction in the condition, e.g., cognitive impairment, ordelay of cognitive impairment and/or cognitive improvement in the adultmammal. For example, an effective dose is the dose that, whenadministered for a suitable period of time, will slow e.g., by about 20%or more, e.g., by 30% or more, by 40% or more, or by 50% or more, insome instances by 60% or more, by 70% or more, by 80% or more, or by 90%or more. For example, will halt, cognitive decline in a patientsuffering from natural aging or an aging-associated disorder. In someinstances, an effective amount or dose of blood product will not onlyslow or halt the progression of the disease condition but will alsoinduce the reversal of the condition, i.e., will cause an improvement incognitive ability. For example, in some instances, an effective amountis the amount that when administered for a suitable period of time,usually at least about one week, and maybe about two weeks, or more, upto a person of about 3 weeks, 4 weeks, 8 weeks, or longer will improvethe cognitive abilities of an individual suffering from anaging-associated cognitive impairment by, for example, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, in some instances 6-fold, 7-fold, 8-fold,9-fold, or 10-fold or more relative to cognition prior to administrationof the blood product or fraction. In some instances, an effective amountor dose of active agent will not only slow or halt the progression ofthe disease condition but will also induce the reversal of thecondition, i.e., will cause an improvement in cognitive function. Forexample, in some instances, an effective amount is the amount that whenadministered for a suitable period of time, will improve the symptoms anindividual suffering from cognitive decline or impairment, for example1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in some instances 6-fold,7-fold, 8-fold, 9-fold, or 10-fold or more relative to untreatedindividuals prior to administration of the agent.

In other embodiments, the blood plasma fraction or Plasma Fraction isadministered in accordance with one or more dosing regimens described inU.S. Patent Application No. 62/490,519, which is herein incorporated byreference in its entirety. As such, an embodiment of the inventionincludes treating a subject diagnosed with a cognitive impairment byadministering to the subject an effective amount of blood plasma orPlasma Fraction wherein the blood plasma or Plasma Fraction isadministered in a manner resulting in improved cognitive function orneurogenesis after the mean or median half-life of the blood plasmaproteins or Plasma Fraction proteins been reached, relative to the mostrecent administered dose (referred to as “Pulsed Dosing” or “PulseDosed” herein). Another embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof at least two consecutive days and monitoring the subject for improvedcognitive function at least 3 days after the date of lastadministration. A further embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive daysand monitoring the subject for improved cognitive function at least 3days after the date of last administration. Yet another embodiment ofthe invention includes administering the blood plasma or Plasma Fractionvia a dosing regimen of at least 2 consecutive days and after the dateof last administration, monitoring for cognitive improvement beyond whenthe average half-life of the proteins in the blood plasma or PlasmaFraction has been reached. Another embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof 2 to 14 non-consecutive days wherein each gap between doses may bebetween 0-3 days each. In some instances, Pulsed Dosing in accordancewith the invention includes administration of a first set of doses,e.g., as described above, followed by a period of no dosing, e.g., a“dosing-free period”, which in turn is followed by administration ofanother dose or set of doses. The duration of this “dosing-free” period,may vary, but in some embodiments, is 7 days or longer, such as 10 daysor longer, including 14 days or longer, wherein some instances thedosing-free period ranges from 15 to 365 days, such as 30 to 90 days andincluding 30 to 60 days. As such, embodiments of the methods includenon-chronic (i.e., non-continuous) dosing, e.g., non-chronicadministration of a blood plasma product. In some embodiments, thepattern of Pulsed Dosing followed by a dosing-free period is repeatedfor a number of times, as desired, where in some instances this patternis continued for 1 year or longer, such as 2 years or longer, up to andincluding the life of the subject. Another embodiment of the inventionincludes administering the blood plasma or Plasma Fraction via a dosingregimen of 5 consecutive days, with a dosing-free period of 2-3 days,followed by administration for 2-14 consecutive days.

Biochemically, by an “effective amount” or “effective dose” of activeagent is meant an amount of active agent that will inhibit, antagonize,decrease, reduce, or suppress by about 20% or more, e.g., by 30% ormore, by 40% or more, or by 50% or more, in some instances by 60% ormore, by 70% or more, by 80% or more, or by 90% or more, in some casesby about 100%, i.e., to negligible amounts, and in some instances,reverse the progression of the cognitive impairment or age-associateddementia.

11. Plasma Protein Fraction

In practicing methods of the invention, a plasma fraction isadministered to the subject. In an embodiment, the Plasma Fraction isplasma protein fraction (PPF). In additional embodiments, the PPF isselected from the Commercial PPF Preparations.

In another embodiment, the PPF is comprised of 88% normal human albumin,12% alpha and beta globulins and not more than 1% gamma globulin asdetermined by electrophoresis. Embodiments of this embodiment used inpracticing methods of the invention include, for example, thisembodiment as a 5% solution of PPF buffered with sodium carbonate andstabilized with 0.004 M sodium caprylate and 0.004 M acetyltryptophan.Additional formulations, including those modifying the percentage of PPF(e.g. about 1% to about 10%, about 10% to about 20%, about 20% to 25%,about 25% to 30%) in solution as well as the concentrations of solventand stabilizers may be utilized in practicing methods of the invention.

12. Plasma Fractions of Specific Donor Age

An embodiment of invention includes administering a blood plasmafraction or a Plasma Fraction derived from the plasma of individuals ofcertain age ranges. Additional embodiments include administering aplasma protein fraction derived from the plasma of individuals ofcertain age ranges. An embodiment includes administering a PPF or a HASwhich has been derived from the plasma of young individuals. In anotherembodiment of the invention the young individuals are of a singlespecific age or a specific age range. In yet another embodiment, theaverage age of the donors is less than that of the subject or less thanthe average age of the subjects being treated.

Certain embodiments of the invention include pooling blood or bloodplasma from individuals of specific age ranges and fractionating theblood plasma as described above to attain a plasma protein fractionproduct such as PPF or HAS. In an alternate embodiment of the invention,the plasma protein fraction or specific plasma protein fraction isattained from specific individuals fitting a specified age range. Inanother embodiment of the invention, the blood plasma fraction, PlasmaFraction, or specific plasma protein fraction product is attained from apool of young individuals, of which “young” may be determined bychronologic or biologic age as described above, and the age(s) of theindividuals may be a specific age or age range.

13. Indications

The subject methods and plasma-comprising blood products and fractionsfind use in treating, including preventing, aging-associated conditions,such as impairments in the cognitive ability of individuals, e.g.,cognitive disorders, including (but not limited to) age-associateddementia, immunological conditions, cancer, and physical and functionaldecline. Individuals suffering from or at risk of developing anaging-associated cognitive impairment that will benefit from treatmentwith the subject plasma-comprising blood product, e.g., by the methodsdisclosed herein, include individuals that are about 50 years old orolder, e.g., 60 years old or older, 70 years old or older, 80 years oldor older, 90 years old or older, and 100 years old or older, i.e.,between the age of about 50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or about 100 years old, and are suffering from cognitiveimpairment associated with natural aging process, e.g., mild cognitiveimpairment (M.C.I.); and individuals that are about 50 years old orolder, e.g., 60 years old or older, 70 years old or older, 80 years oldor older, 90 years old or older, and usually no older than 100 yearsold, i.e., between the ages of about 50 and 90, e.g., 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or about 100 years old, that have not yet begunto show symptoms of cognitive impairment. Examples of cognitiveimpairments that are due to natural aging include the following:

A. Mild cognitive impairment (M.C.I.) is a modest disruption ofcognition that manifests as problems with memory or other mentalfunctions such as planning, following instructions, or making decisionsthat have worsened over time while overall mental function and dailyactivities are not impaired. Thus, although significant neuronal deathdoes not typically occur, neurons in the aging brain are vulnerable tosub-lethal age-related alterations in structure, synaptic integrity, andmolecular processing at the synapse, all of which impair cognitivefunction.

Individuals suffering from or at risk of developing an aging-associatedcognitive impairment that will benefit from treatment with the subjectplasma-comprising blood product or fraction, e.g., by the methodsdisclosed herein, also include individuals of any age that are sufferingfrom a cognitive impairment due to an aging-associated disorder; andindividuals of any age that have been diagnosed with an aging-associateddisorder that is typically accompanied by cognitive impairment, wherethe individual has not yet begun to present with symptoms of cognitiveimpairment. Examples of such aging-associated disorders include thefollowing:

B. Alzheimer's disease. Alzheimer's disease is a progressive, inexorableloss of cognitive function associated with an excessive number of senileplaques in the cerebral cortex and subcortical gray matter, which alsocontains b-amyloid and neurofibrillary tangles consisting of tauprotein. The common form affects persons >60 yr old, and its incidenceincreases as age advances. It accounts for more than 65% of thedementias in the elderly.

The cause of Alzheimer's disease is not known. The disease runs infamilies in about 15 to 20% of cases. The remaining, so-called sporadiccases have some genetic determinants. The disease has an autosomaldominant genetic pattern in most early-onset and some late-onset casesbut a variable late-life penetrance. Environmental factors are the focusof active investigation.

In the course of the disease, synapses, and ultimately neurons are lostwithin the cerebral cortex, hippocampus, and subcortical structures(including selective cell loss in the nucleus basalis of Meynert), locuscoeruleus, and nucleus raphae dorsalis. Cerebral glucose use andperfusion is reduced in some areas of the brain (parietal lobe andtemporal cortices in early-stage disease, prefrontal cortex inlate-stage disease). Neuritic or senile plaques (composed of neurites,astrocytes, and glial cells around an amyloid core) and neurofibrillarytangles (composed of paired helical filaments) play a role in thepathogenesis of Alzheimer's disease. Senile plaques and neurofibrillarytangles occur with normal aging, but they are much more prevalent inpersons with Alzheimer's disease.

C. Parkinson's Disease. Parkinson's Disease (PD) is an idiopathic,slowly progressive, degenerative CNS disorder characterized by slow anddecreased movement, muscular rigidity, resting tremor, and posturalinstability. Originally considered primarily a motor disorder, PD is nowrecognized to also affect cognition, behavior, sleep, autonomicfunction, and sensory function. The most common cognitive impairmentsinclude an impairment in attention and concentration, working memory,executive function, producing language, and visuospatial function.

In primary Parkinson's disease, the pigmented neurons of the substantianigra, locus coeruleus, and other brain stem dopaminergic cell groupsare lost. The cause is not known. The loss of substantia nigra neurons,which project to the caudate nucleus and putamen, results in depletionof the neurotransmitter dopamine in these areas. Onset is generallyafter age 40, with increasing incidence in older age groups.

Secondary parkinsonism results from loss of or interference with theaction of dopamine in the basal ganglia due to other idiopathicdegenerative diseases, drugs, or exogenous toxins. The most common causeof secondary parkinsonism is ingestion of antipsychotic drugs orreserpine, which produce parkinsonism by blocking dopamine receptors.Less common causes include carbon monoxide or manganese poisoning,hydrocephalus, structural lesions (tumors, infarcts affecting themidbrain or basal ganglia), subdural hematoma, and degenerativedisorders, including striatonigral degeneration.

D. Frontotemporal dementia. Frontotemporal dementia (FTD) is a conditionresulting from the progressive deterioration of the frontal lobe of thebrain. Over time, the degeneration may advance to the temporal lobe.Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for20% of pre-senile dementia cases. Symptoms are classified into threegroups based on the functions of the frontal and temporal lobesaffected:

Behavioral variant FTD (bvFTD), with symptoms include lethargy andaspontaneity on the one hand, and disinhibition on the other;progressive nonfluent aphasia (PNFA), in which a breakdown in speechfluency due to articulation difficulty, phonological and/or syntacticerrors is observed but word comprehension is preserved; and semanticdementia (SD), in which patients remain fluent with normal phonology andsyntax but have increasing difficulty with naming and wordcomprehension. Other cognitive symptoms common to all FTD patientsinclude an impairment in executive function and ability to focus. Othercognitive abilities, including perception, spatial skills, memory andpraxis typically remain intact. FTD can be diagnosed by observation ofreveal frontal lobe and/or anterior temporal lobe atrophy in structuralMRI scans.

A number of forms of FTD exist, any of which may be treated or preventedusing the subject methods and compositions. For example, one form offrontotemporal dementia is Semantic Dementia (SD). SD is characterizedby a loss of semantic memory in both the verbal and non-verbal domains.SD patients often present with the complaint of word-findingdifficulties. Clinical signs include fluent aphasia, anomia, impairedcomprehension of word meaning, and associative visual agnosia (theinability to match semantically related pictures or objects). As thedisease progresses, behavioral and personality changes are often seensimilar to those seen in frontotemporal dementia although cases havebeen described of ‘pure’ semantic dementia with few late behavioralsymptoms. Structural MRI imaging shows a characteristic pattern ofatrophy in the temporal lobes (predominantly on the left), with inferiorgreater than superior involvement and anterior temporal lobe atrophygreater than posterior.

As another example, another form of frontotemporal dementia is Pick'sdisease (PiD, also PcD). A defining characteristic of the disease isbuild-up of tau proteins in neurons, accumulating into silver-staining,spherical aggregations known as “Pick bodies.” Symptoms include loss ofspeech (aphasia) and dementia. Patients with orbitofrontal dysfunctioncan become aggressive and socially inappropriate. They may steal ordemonstrate obsessive or repetitive stereotyped behaviors. Patients withdorsomedial or dorsolateral frontal dysfunction may demonstrate a lackof concern, apathy, or decreased spontaneity. Patients can demonstratean absence of self-monitoring, abnormal self-awareness, and an inabilityto appreciate meaning. Patients with gray matter loss in the bilateralposterolateral orbitofrontal cortex and right anterior insula maydemonstrate changes in eating behaviors, such as a pathologic sweettooth. Patients with more focal gray matter loss in the anterolateralorbitofrontal cortex may develop hyperphagia. While some of the symptomscan initially be alleviated, the disease progresses and patients oftendie within two to ten years.

E. Huntington's disease. Huntington's disease (HD) is a hereditaryprogressive neurodegenerative disorder characterized by the developmentof emotional, behavioral, and psychiatric abnormalities; loss ofintellectual or cognitive functioning; and movement abnormalities (motordisturbances). The classic signs of HD include the development ofchorea—involuntary, rapid, irregular, jerky movements that may affectthe face, arms, legs, or trunk—as well as cognitive decline includingthe gradual loss of thought processing and acquired intellectualabilities. There may be impairment of memory, abstract thinking, andjudgment; improper perceptions of time, place, or identity(disorientation); increased agitation; and personality changes(personality disintegration). Although symptoms typically become evidentduring the fourth or fifth decades of life, the age at onset is variableand ranges from early childhood to late adulthood (e.g., 70s or 80s).

HD is transmitted within families as an autosomal dominant trait. Thedisorder occurs as the result of abnormally long sequences or “repeats”of coded instructions within a gene on chromosome 4 (4p16.3). Theprogressive loss of nervous system function associated with HD resultsfrom loss of neurons in certain areas of the brain, including the basalganglia and cerebral cortex.

F. Amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) isa rapidly progressive, invariably fatal, neurological disease thatattacks motor neurons. Muscular weakness and atrophy and signs ofanterior horn cell dysfunction are initially noted most often in thehands and less often in the feet. The site of onset is random, andprogression is asymmetric. Cramps are common and may precede weakness.Rarely, a patient survives 30 years; 50% die within 3 years of onset,20% live 5 years, and 10% live 10 years.

Diagnostic features include onset during middle or late adult life andprogressive, generalized motor involvement without sensoryabnormalities. Nerve conduction velocities are normal until late in thedisease. Recent studies have documented the presentation of cognitiveimpairments as well, particularly a reduction in immediate verbalmemory, visual memory, language, and executive function.

A decrease in cell body area, number of synapses and total synapticlength has been reported in even normal-appearing neurons of the ALSpatients. It has been suggested that when the plasticity of the activezone reaches its limit, a continuing loss of synapses can lead tofunctional impairment. Promoting the formation or new synapses orpreventing synapse loss may maintain neuron function in these patients.

G. Multiple Sclerosis. Multiple Sclerosis (MS) is characterized byvarious symptoms and signs of CNS dysfunction, with remissions andrecurring exacerbations. The most common presenting symptoms areparesthesias in one or more extremities, in the trunk, or on one side ofthe face; weakness or clumsiness of a leg or hand; or visualdisturbances, e.g., partial blindness and pain in one eye (retrobulbaroptic neuritis), dimness of vision, or scotomas. Common cognitiveimpairments include impairments in memory (acquiring, retaining, andretrieving new information), attention and concentration (particularlydivided attention), information processing, executive functions,visuospatial functions, and verbal fluency. Common early symptoms areocular palsy resulting in double vision (diplopia), transient weaknessof one or more extremities, slight stiffness or unusual fatigability ofa limb, minor gait disturbances, difficulty with bladder control,vertigo, and mild emotional disturbances; all indicate scattered CNSinvolvement and often occur months or years before the disease isrecognized. Excess heat may accentuate symptoms and signs.

The course is highly varied, unpredictable, and, in most patients,remittent. At first, months or years of remission may separate episodes,especially when the disease begins with retrobulbar optic neuritis.However, some patients have frequent attacks and are rapidlyincapacitated; for a few the course can be rapidly progressive.

H. Glaucoma. Glaucoma is a common neurodegenerative disease that affectsretinal ganglion cells (RGCs). Evidence supports the existence ofcompartmentalized degeneration programs in synapses and dendrites,including in RGCs. Recent evidence also indicates a correlation betweencognitive impairment in older adults and glaucoma (Yochim B P, et al.Prevalence of cognitive impairment, depression, and anxiety symptomsamong older adults with glaucoma. J Glaucoma. 2012; 21(4):250-254).

I. Myotonic dystrophy. Myotonic dystrophy (DM) is an autosomal dominantmultisystem disorder characterized by dystrophic muscle weakness andmyotonia. The molecular defect is an expanded trinucleotide (CTG) repeatin the 3′ untranslated region of the myotoninprotein kinase gene onchromosome 19q. Symptoms can occur at any age, and the range of clinicalseverity is broad. Myotonia is prominent in the hand muscles, and ptosisis common even in mild cases. In severe cases, marked peripheralmuscular weakness occurs, often with cataracts, premature balding,hatchet facies, cardiac arrhythmias, testicular atrophy, and endocrineabnormalities (e.g., diabetes mellitus). Mental retardation is common insevere congenital forms, while an aging-related decline of frontal andtemporal cognitive functions, particularly language and executivefunctions, is observed in milder adult forms of the disorder. Severelyaffected persons die by their early 50s.

J. Dementia. Dementia describes a class of disorders having symptomsaffecting thinking and social abilities severely enough to interferewith daily functioning. Other instances of dementia in addition to thedementia observed in later stages of the aging-associated disordersdiscussed above include vascular dementia, and dementia with Lewybodies, described below.

In vascular dementia, or “multi-infarct dementia”, cognitive impairmentis caused by problems in supply of blood to the brain, typically by aseries of minor strokes, or sometimes, one large stroke preceded orfollowed by other smaller strokes. Vascular lesions can be the result ofdiffuse cerebrovascular disease, such as small vessel disease, or focallesions, or both. Patients suffering from vascular dementia present withcognitive impairment, acutely or subacutely, after an acutecerebrovascular event, after which progressive cognitive decline isobserved. Cognitive impairments are similar to those observed inAlzheimer's disease, including impairments in language, memory, complexvisual processing, or executive function, although the related changesin the brain are not due to AD pathology but to chronic reduced bloodflow in the brain, eventually resulting in dementia. Single photonemission computed tomography (SPECT) and positron emission tomography(PET) neuroimaging may be used to confirm a diagnosis of multi-infarctdementia in conjunction with evaluations involving mental statusexamination.

Dementia with Lewy bodies (DLB, also known under a variety of othernames including Lewy body dementia, diffuse Lewy body disease, corticalLewy body disease, and senile dementia of Lewy type) is a type ofdementia characterized anatomically by the presence of Lewy bodies(clumps of alpha-synuclein and ubiquitin protein) in neurons, detectablein post mortem brain histology. Its primary feature is cognitivedecline, particularly of executive functioning. Alertness and short termmemory will rise and fall.

Persistent or recurring visual hallucinations with vivid and detailedpictures are often an early diagnostic symptom. DLB it is often confusedin its early stages with Alzheimer's disease and/or vascular dementia,although, where Alzheimer's disease usually begins quite gradually, DLBoften has a rapid or acute onset. DLB symptoms also include motorsymptoms similar to those of Parkinson's. DLB is distinguished from thedementia that sometimes occurs in Parkinson's disease by the time framein which dementia symptoms appear relative to Parkinson symptoms.Parkinson's disease with dementia (POD) would be the diagnosis whendementia onset is more than a year after the onset of Parkinson's. DLBis diagnosed when cognitive symptoms begin at the same time or within ayear of Parkinson symptoms.

K. Progressive supranuclear palsy. Progressive supranuclear palsy (PSP)is a brain disorder that causes serious and progressive problems withcontrol of gait and balance, along with complex eye movement andthinking problems. One of the classic signs of the disease is aninability to aim the eyes properly, which occurs because of lesions inthe area of the brain that coordinates eye movements. Some individualsdescribe this effect as a blurring. Affected individuals often showalterations of mood and behavior, including depression and apathy aswell as progressive mild dementia. The disorder's long name indicatesthat the disease begins slowly and continues to get worse (progressive),and causes weakness (palsy) by damaging certain parts of the brain abovepea-sized structures called nuclei that control eye movements(supranuclear). PSP was first described as a distinct disorder in 1964,when three scientists published a paper that distinguished the conditionfrom Parkinson's disease. It is sometimes referred to asSteele-Richardson-Olszewski syndrome, reflecting the combined names ofthe scientists who defined the disorder. Although PSP gets progressivelyworse, no one dies from PSP itself.

L. Ataxia. People with ataxia have problems with coordination becauseparts of the nervous system that control movement and balance areaffected. Ataxia may affect the fingers, hands, arms, legs, body,speech, and eye movements. The word ataxia is often used to describe asymptom of incoordination which can be associated with infections,injuries, other diseases, or degenerative changes in the central nervoussystem. Ataxia is also used to denote a group of specific degenerativediseases of the nervous system called the hereditary and sporadicataxias which are the National Ataxia Foundation's primary emphases.

M. Multiple-system atrophy. Multiple-system atrophy (MSA) is adegenerative neurological disorder. MSA is associated with thedegeneration of nerve cells in specific areas of the brain. This celldegeneration causes problems with movement, balance, and other autonomicfunctions of the body such as bladder control or blood-pressureregulation.

The cause of MSA is unknown and no specific risk factors have beenidentified. Around 55% of cases occur in men, with typical age of onsetin the late 50s to early 60s. MSA often presents with some of the samesymptoms as Parkinson's disease. However, MSA patients generally showminimal if any response to the dopamine medications used forParkinson's.

N. Frailty. Frailty Syndrome (“Frailty”) is a geriatric syndromecharacterized by functional and physical decline including decreasedmobility, muscle weakness, physical slowness, poor endurance, lowphysical activity, malnourishment, and involuntary weight loss. Suchdecline is often accompanied and a consequence of diseases such ascognitive dysfunction and cancer. However, Frailty can occur evenwithout disease. Individuals suffering from Frailty have an increasedrisk of negative prognosis from fractures, accidental falls, disability,comorbidity, and premature mortality. (C. Buigues, et al. Effect of aPrebiotic Formulation on Frailty Syndrome: A Randomized, Double-BlindClinical Trial, Int. J. Mol. Sci. 2016, 17, 932). Additionally,individuals suffering from Frailty have an increased incidence of higherhealth care expenditure. (Id.)

Common symptoms of Frailty can be determined by certain types of tests.For example, unintentional weight loss involves a loss of at least 10lbs. or greater than 5% of body weight in the preceding year; muscleweakness can be determined by reduced grip strength in the lowest 20% atbaseline (adjusted for gender and BMI); physical slowness can be basedon the time needed to walk a distance of 15 feet; poor endurance can bedetermined by the individual's self-reporting of exhaustion; and lowphysical activity can be measured using a standardized questionnaire.(Z. Palace et al., The Frailty Syndrome, Today's Geriatric Medicine7(1), at 18 (2014)).

In some embodiments, the subject methods and compositions find use inslowing the progression of aging-associated cognitive impairment. Inother words, cognitive abilities in the individual will decline moreslowly following treatment by the disclosed methods than prior to or inthe absence of treatment by the disclosed methods. In some suchinstances, the subject methods of treatment include measuring theprogression of cognitive decline after treatment, and determining thatthe progression of cognitive decline is reduced. In some such instances,the determination is made by comparing to a reference, e.g., the rate ofcognitive decline in the individual prior to treatment, e.g., asdetermined by measuring cognition prior at two or more time points priorto administration of the subject blood product.

The subject methods and compositions also find use in stabilizing thecognitive abilities of an individual, e.g., an individual suffering fromaging-associated cognitive decline or an individual at risk of sufferingfrom aging-associated cognitive decline. For example, the individual maydemonstrate some aging-associated cognitive impairment, and progressionof cognitive impairment observed prior to treatment with the disclosedmethods will be halted following treatment by the disclosed methods. Asanother example, the individual may be at risk for developing anaging-associated cognitive decline (e.g., the individual may be aged 50years old or older, or may have been diagnosed with an aging-associateddisorder), and the cognitive abilities of the individual aresubstantially unchanged, i.e., no cognitive decline can be detected,following treatment by the disclosed methods as compared to prior totreatment with the disclosed methods.

The subject methods and compositions also find use in reducing cognitiveimpairment in an individual suffering from an aging-associated cognitiveimpairment. In other words, cognitive ability is improved in theindividual following treatment by the subject methods. For example, thecognitive ability in the individual is increased, e.g., by 2-fold ormore, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more,30-fold or more, or 40-fold or more, including 50-fold or more, 60-foldor more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-foldor more, following treatment by the subject methods relative to thecognitive ability that is observed in the individual prior to treatmentby the subject methods. In some instances, treatment by the subjectmethods and compositions restores the cognitive ability in theindividual suffering from aging-associated cognitive decline, e.g., totheir level when the individual was about 40 years old or less. In otherwords, cognitive impairment is abrogated.

14. Methods of Diagnosing and Monitoring for Improvement ofNeurocognitive-Associated Disease

In some instances, among the variety of methods to diagnose and monitordisease progression and improvement in neurocognitive-associateddisease, the following types of assessments are used alone or incombination with subjects suffering from neurodegenerative disease, asdesired. The following types of methods are presented as examples andare not limited to the recited methods. Any convenient methods tomonitor disease may be used in practicing the invention, as desired.Those methods are also contemplated by the methods of the invention.

A. General Cognition

Embodiments of the methods of the invention further comprise methods ofmonitoring the effect of a medication or treatment on a subject fortreating cognitive impairment and/or age-related dementia, the methodcomprising comparing cognitive function before and after treatment.Those having ordinary skill in the art recognize that there arewell-known methods of evaluating cognitive function. For example, andnot by way of limitation, the method may comprise evaluation ofcognitive function based on medical history, family history, physicaland neurological examinations by clinicians who specialize dementia andcognitive function, laboratory tests, and neuropsychological assessment.Additional embodiments which are contemplated by the invention include:the assessment of consciousness, such as using the Glasgow Coma Scale(EMV); mental status examination, including the abbreviated mental testscore (AMTS) or mini-mental state examination (MMSE) (Folstein et al.,J. Psychiatr. Res 1975; 12:1289-198); global assessment of higherfunctions; estimation of intracranial pressure such as by fundoscopy.

In one embodiment, examinations of peripheral nervous system may be usedto evaluate cognitive function, including any one of the followings:sense of smell, visual fields and acuity, eye movements and pupils(sympathetic and parasympathetic), sensory function of face, strength offacial and shoulder girdle muscles, hearing, taste, pharyngeal movementand reflex, tongue movements, which can be tested individually (e.g. thevisual acuity can be tested by a Snellen chart; a reflex hammer usedtesting reflexes including masseter, biceps and triceps tendon, kneetendon, ankle jerk and plantar (i.e. Babinski sign); Muscle strengthoften on the MRC scale 1 to 5; Muscle tone and signs of rigidity.

15. Reagents, Devices, and Kits

Also provided are reagents, devices, and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devices,and kits thereof may vary greatly.

Reagents and devices of interest include those mentioned above withrespect to the methods of preparing plasma-comprising blood product fortransfusion into a subject in need hereof, for example, anti-coagulants,cryopreservatives, buffers, isotonic solutions, etc.

Kits may also comprise blood collection bags, tubing, needles,centrifugation tubes, and the like. In yet other embodiments, kits asdescribed herein include two or more containers of blood plasma productsuch as plasma protein fraction, such as three or more, four or more,five or more, including six or more containers of blood plasma product.In some instances, the number of distinct containers of blood plasmaproduct in the kit may be 9 or more, 12 or more, 15 or more, 18 or more,21 or more, 24 or more 30 or more, including 36 or more, e.g., 48 ormore. Each container may have associated therewith identifyinginformation which includes various data about the blood plasma productcontained therein, which identifying information may include one or moreof the age of the donor of the blood plasma product, processing detailsregarding the blood plasma product, e.g., whether the plasma product wasprocessed to remove proteins above an average molecule weight (such asdescribed above), blood type details, etc. In some instances, eachcontainer in the kit includes identifying information about the bloodplasma contained therein, and the identifying information includesinformation about the donor age of the blood plasma product, e.g., theidentifying information provides confirming age-related data of theblood plasma product donor (where such identifying information may bethe age of the donor at the time of harvest). In some instances, eachcontainer of the kit contains a blood plasma product from a donor ofsubstantially the same age, i.e., all of the containers include productfrom donors that are substantially the same, if not the same, age. Bysubstantially the same age is meant that the various donors from whichthe blood plasma products of the kits are obtained differ in each, insome instances, by 5 years or less, such as 4 years or less, e.g., 3years or less, including 2 years or less, such as 1 year or less, e.g.,9 months or less, 6 months or less, 3 months or less, including 1 monthor less. The identifying information can be present on any convenientcomponent of the container, such as a label, an RFID chip, etc. Theidentifying information may be human readable, computer readable, etc.,as desired. The containers may have any convenient configuration. Whilethe volume of the containers may vary, in some instances the volumesrange from 10 ml to 5000 mL, such as 25 mL to 2500 mL, e.g., 50 ml to1000 mL, including 100 mL to 500 mL. The containers may be rigid orflexible, and may be fabricated from any convenient material, e.g.,polymeric materials, including medical grade plastic materials. In someinstances, the containers have a bag or pouch configuration. In additionto the containers, such kits may further include administration devices,e.g., as described above. The components of such kits may be provided inany suitable packaging, e.g., a box or analogous structure, configuredto hold the containers and other kit components.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, portable flash drive, etc., on which the informationhas been recorded. Yet another means that may be present is a websiteaddress which may be used via the internet to access the information ata removed site. Any convenient means may be present in the kits.

16. Experimental Procedures

The following examples are put forth to provide those of ordinary skillin the art with a complete disclosure and description of how to make anduse the present invention, and are not intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis degrees Centigrade, and pressure is at near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995; ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Precedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and Clontech.

A. Materials and Reagents.

USP saline was purchased from Hospira (Lake Forest, Ill.). Injectionswere performed with 27.5 G or 30 G needles, at a volume of 150 μL perinjection. Commercially-available PPF (“PPF1”) such as those CommercialPPF Preparations described above in 5% solution were stored at 4° C.Commercially-available HAS (“HAS1”) such as those Commercial HASPreparations described above in 5% solution were stored at 4° C.

B. Animal Supply and Husbandry.

The mouse strains NOD.CB17-Prkdcscid/NcrCrl (“NODscid,” Strain Code 394,Charles River, Mass.) (Bosma, M. et al., The scid mouse mutant. 137 CurrTop Microbiol Immunol 197 (1988)) and NOD scid gamma (“NSG,” Strain Code005557, The Jackson Laboratory, Bar Harbor, ME) were used. Each mousewas ear punched to designate a unique identification number. All micewere individually housed under specific pathogen-free conditions under a12-hour light, 12-hour dark cycle, and all animal handling and use wasin accordance with IACUC approved standard guidelines.

C. Administration.

Unless described differently below, NSG and NODscid mice were injectedwith USP saline, 5% PPPF1, or 5% HAS1 twice weekly via intravenous tailvein injection (150 μL per injection) for up to 6 months.

D. Open Field

Open field tests were utilized to determine exploratory behavior of thesubject mice. The open field test is an empty test arena, usually roundor square. The mouse is placed inside a 50 cm×50 cm open filed arena for15 minutes and the level of the mouse's activity is measured. Rearingtime was measured by tracking the duration the forepaws were on thewalls of the box. Total distance covered and velocity was also measuredfor duration of the test. CleverSys TopScan V3.0 (Reston, Va.) was usedto track mouse behavior in open field. Open field chambers wereconstructed by CleverSys.

E. Y-Maze

Mice were allowed to explore two arms of a Y-maze (start+familiar) for 5minutes. One hour later, mice were allowed to explore all three arms,and total time and number of entries in the arms were recorded.

F. Barnes Maze

Mice were trained on four consecutive days in a modified Barnes maze andgiven a maximum of 120 seconds to find the escape hole. (See Barnes, C.A., Memory deficits associated with senescence: A neurophysiological andbehavioral study in the rat, J. COMPARATIVE AND PHYSIOLOGICALPSYCHOLOGY, 93(1): 74-104 (1979); and for the modified maze, Faizi, M.et al., Thy1-hAPP(Lond/Swe+) mouse model of Alzheimer's disease displaysbroad behavioral deficits in sensorimotor, cognitive and socialfunction., BRAIN BEHAV. 2(2): 142-54, (2012)). The escape hole remainedthe same for four trials on a training day, but changed between trainingdays. The latency to the escape hole was recorded for each mouse cohorton four separate training days.

G. DCX- and Ki67-Positive Cells

Doublecortin (DCX) is a microtubule□associated protein that is expressedby neuronal precursor cells. It is also expressed by immature neurons inembryonic and adult cortical structures. When they are activelydividing, neuronal precursor cells express DCX. The proteindownregulates after two weeks. Because of this association, it is usefulas a marker of neurogenesis.

Brain tissue processing and immunohistochemistry was performed onfree-floating sections well-described techniques (Luo, J. et al.Glia-dependent TGF-b signaling, acting independently of the TH17pathway, is critical for initiation of murine autoimmuneencephalomyelitis. J. CLIN. INVEST. 117, 3306-3315 (2007)). Mice wereanesthetized and perfused with 0.9% saline. Brains were removed andsubsequently fixed with phosphate-buffered 4% paraformaldehyde, pH 7.4,at 4° C. before sunk through 30% sucrose for cryoprotection. Brains weresubsequently sectioned at 30 μm with a cryomicrotome at −22° C. Sectionswere stored in cyroprotective medium. The primary antibody used was goatanti-Dcx (Santa Cruz Biotechnology at 1:500 for twice weekly dosingexperiments or 1:200 in the three times per week dosing experiments) orrabbit anti-Ki67 (1:500 Abcam). Primary antibody staining was revealedusing biotinylated secondary antibodies and the ABCkit (Vector) withdiaminobenzidine (DAB, Sigma-Aldrich) or fluorescence-conjugatedsecondary antibodies. To estimate the total number of Dcx-positive cellsper dentate gyrus, immunopositive cells in the granule cell andsubgranular cell layer of the dentate gyms were counted in three coronalhemibrain sections through the hippocampus and averaged.

H. Barnes Maze Test on Aged NSG Mice Treated with Young Plasma, EffluentI, or Effluent II/III

Aged NSG mice (aged 12 months), were separated into several groups (allof n=14), and received 150 μL saline, young plasma, Effluent I, orEffluent II/III by tail vein injection prior to initiation of behaviortests. Each separate group was separated into 3 cohorts with each cohortinitiated with behavior tests on a different week.

I. Barnes Maze and Cell Survival (BrdU Staining) in Aged NSG MiceTreated Three Times Per Week with Young Plasma or PPF1

Aged (12 months) male NSG mice were treated intravenously via tail veininjection with 150 μL of clarified young human plasma (young plasma),PPPF1, or saline three times per week for four weeks. The regimen waschanged to twice per week during weeks 5 and 6, which were thebehavioral testing weeks.

Prior to treatment, the mice were divided into three cohorts of 13-15mice each. Each cohort underwent five days of BrdU injectionsintraperitoneally (i.p.) prior to the start of treatment of youngplasma, PPPF1, or saline as described above.

During weeks 5 and 6, behavioral testing was performed, and latency totarget hole was measured for each mouse in Barnes Maze testing. Eachtesting session lasted for a maximum of 120 seconds. The event offinding the target hole was recorded using software that determined whenthe nose of the mouse entered the area defined as the target hole.

At the end of behavioral testing, the animals were sacrificed, and sixsections per hippocampus were quantified using brightfield microscope todetermine presence of BrdU positive cells within the granule cell layerof the dentate gyms. As representative sections throughout the differentregions of the hippocampus, the average number of BrdU positive cellswere multiplied by 72, which was the total number of sections for eachanimal's hippocampus, in order to give an estimate of the total numberof BrdU positive cells.

J. Neurosphere and Cortex Culture Assays

1. Tuj1 and DAPI Staining

Mouse C57 E14,15 cortices (Lonza: M-CX-300) were suspended in 12 ml ofneural basal media supplemented with B27, 2 mM Glutamax (Sigma-Aldrich).200 μL was added to each well of a 96-well plate pre-coated withcollagen I (Corning, Inc.). After 16 hours, plating media was replacedwith pre-warmed (37° C.) control media (Neural basal media with B27, 2mM Glutamax (Gibco). On day 4 in vitro (“days in vitro”, or “DIV”),culture media was replaced with fresh control media, control media and10% PPPF1, control media and 10% HAS1, vehicle and 10% PPPF1, or vehicleand 10% HAS1. Cultures were maintained for 21 days with 75% of mediachanged to fresh media every 3 days. At 21 DIV, cultures were washed 3×with PBS then fixed with 4% Paraformaldehyde for 20 minutes at roomtemperature (RT). After fixation, cultures were washed 2× with PBS thenpermeabilized with 0.1% Triton X100 for 5-20 minutes. Afterpermeabilization, cultures were blocked with 3% bovine serum albumin(Sigma-Aldrich) for 60 minutes at RT. After 60 minutes, blockingsolution was aspirated and cultures were labeled with anti-Tuj1 antibody(AbCam-1:500) at 4° C. overnight. After labeling, cultures were washed3× with PBS +0.1% BSA then stained with A647-conjugated Donkeyanti-mouse antibody at 4° C. overnight (1:1000). Cultures were thenwashed 2× with PBS and labeled with Hoechst 33342 (1:1000) for 20 min.Samples were washed 3× with PBS after Hoechst labeling. Twenty-five (25)fields were acquired for each well using 10× magnification using GEInCell Analyzer 2000 (GE Healthcare Life Sciences). Results are shown inFIG. 19.

2. Net Neurite Length

Net neurite length was determined from cultures as described in theprevious section. Neurite analysis was performed using a customalgorithm generated by GE InCell Investigator Developer Toolbox. Resultsfrom control and vehicle treated samples were nearly identical thereforewere combined for statistical analysis. Results are shown in FIG. 20.

3. Cortex Culture Sphere Number and Size; Process Length and Branching

Mouse C57 E14,15 cortices (Lonza: M-CX-300) were suspended in 12 mL ofneurobasal media supplemented with B27, 2 mM Glutamax (Sigma-Aldrich).200 μL was added to each well of a 96-well plate pre-coated withpolylysine and laminin. Four days later, 50% of the media was exchangedwith fresh media and treated with test article (vehicle, PPPF1, or HAS1)to a final concentration of 10%. This was repeated three days later. OnDay 7 of treatment, cells were imaged in phase contrast at 10×magnification with IncuCyte (Ann Arbor, Mich.) and analyzed withstandard “Neurite and Cell-Body” algorithms. Six replicates wereanalyzed with four images taken per replicate. Standard error isdisplayed. Significance is shown for 2 tailed T-test as P<0.5. Resultsare displayed in FIGS. 21 and 22.

4. Sox2 Neurosphere Staining

Mouse C57 E14,15 cortical neurons (Lonza: M-CX-300) were suspended inneurobasal media supplemented with B27, 2 mM Glutamax (Sigma-Aldrich) at100-200K cells/ml. 200 μL was added to each well of a 96-well platepre-coated with collagen I (Corning, Inc.). After 16 hours, platingmedia was replaced with pre-warmed (37° C.) control media (Neurobasalmedia with B27, 2 mM Glutamax (Gibco)). On day 4 in vitro (“days invitro”, or “DIV”), culture media was replaced with fresh control media,control media with HAS vehicle (vehicle), control media and 10% PPPF1,control media and 10% HAS1. Cultures were maintained for 21 days with75% of media changed to fresh media every 3-4 days. At 21 DIV, cultureswere washed 3× with PBS then fixed with 4% Paraformaldehyde for 20minutes at room temperature (RT). After fixation, cultures were washed2× with PBS then permeabilized with 0.1% Triton 100× for 5-20 minutes.After permeabilization, cultures were blocked with 3% bovine serumalbumin (Sigma-Aldrich) for 60 minutes at RT. After 60 minutes, blockingsolution was aspirated and cultures were labeled with anti-Tuj1 antibody(AbCam-1:500) and Rabbit anti SOX2 (AbCam: 1:5000 at 4° C. overnight.After labeling, cultures were washed 3× with PBS +0.1% BSA then stainedwith Donkey anti-mouse-647 (AbCam) and Sheep anti-rabbit-Texas Red at 4°C. overnight (1:1000). Cultures were then washed 2× with PBS and labeledwith Hoechst (1:1000) for 20 min. Samples were washed 3× with PBS afterHoechst labeling. Twenty-five (20 or 25) fields were acquired for eachwell using 10× magnification of InCell Analyzer 2000 (GE Healthcare LifeSciences). Neurosphere and neurite analysis were done using customalgorithm generated by GE InCell Investigator Developer Toolbox. Resultsfrom control and vehicle treated samples were nearly identical thereforewere combined for statistical analysis. Results are displayed in FIG.23.

K. Results of In Vivo Experiments

1. Open Field Test with 3-Month and 13-month-Old NSG Mice

3-month (young) or 13-month-old (old) NSG mice were placed in an OpenField chamber for 15 minutes. The time spent rearing FIG. 1, velocityFIG. 2, and distance FIG. 3 were measured. FIG. 1 shows that13-month-old mice spent less time rearing than 3-month-old mice, butthat PPF1 and HAS1-treated mice were not significantly different fromyoung mice. FIG. 2 shows that saline (control) and PPF1-treated13-month-old mice were significantly slower than 3-month-old mice.However, HAS1-treated mice were significantly faster than saline-treatedmice, and not significantly different from young mice. FIG. 3 shows thatsaline (control) and HAS1-treated old mice had less locomotor activitythan young mice, and PPF1-treated mice covered more distance thansaline-treated mice. All data shown are mean±s.e.m; *P<0.05; **P<0.01;***P<0.001; t-test; n=20, 18, 18, 19. (SAL=saline).

2. Y-Maze Test with 3-Month and 13-Month-Old NSG Mice

Young (3-month-old) and old (13-month-old) NSG mice were tested in thecued Y-maze as a test for memory. FIG. 4 shows that all mice spentsignificantly more time in the novel (N) arm than the familiar (F) arm.FIG. 5 shows that HAS1-treated old mice were significantly impaired intheir memory for the familiar arm compared to young mice, whereasPPF1-treated mice trended towards improved memory for the familiar arm.FIG. 6 shows that saline and PPF1-treated, but not HAS1-treated oldmice, were significantly slower than young mice. FIG. 7 shows thatsaline and PPF1-treated, but not HAS1-treated old mice, covered lessdistance than young mice. All data shown are mean±s.e.m; *P<0.05;**P<0.01; ***P<0.001; Paired t-test; n=20, 18, 18, 19. (SAL=saline).

3. Fear Conditioning Test for Memory with 3-Month and 13-Month-Old NSGMice

Young (3-month-old) and old (13-month-old) NSG mice were tested in thefear conditioning test for memory. FIG. 8A shows that 13-month-old micetrended to spend less time freezing than 3-month-old mice, whereasHAS1-treated mice spent almost as much time freezing as 3-month-oldmice. FIG. 8B shows that in the cued test for memory of the auditorycue, 13-month-old control-treated mice performed the worst and froze theleast amount of time. HAS1-treated mice trended to spend more timefreezing, indicating improved memory for the tone. FIG. 9 shows thequantification of the last 90 seconds of the cued test for memory andshows that HAS1-treated mice trended to spend more time freezing,indicating improved memory. n=20, 16, 17, 19. (SAL=saline).

4. Barnes Maze Test for Spatial Memory with 3-Month and 13-Month-Old NSGMice

Young (3-month-old) and old (13-month-old) NSG mice were tested in theBarnes maze test for spatial memory. FIG. 10A shows that 3-month-oldmice performed the best and had the fastest latency to reach the targethole by the last trial. FIG. 10B shows the quantification of the averageof the last 3 trials which demonstrates that saline- and HAS1-treatedold mice were significantly impaired in their memory of the target holecompared to young mice, but the PPF1-treated mice were not significantlydifferent from young mice. **P<0.01; ***P<0.001; Unpaired t-test; n=20,18, 18, 19. (SAL=saline).

5. Immunostaining with 3-Month and 13-Month-Old NSG Mice

Brain sections were stained for doublecortin (Dcx), a marker for newbornneurons or for Ki67, a marker for proliferating cells in 3-month and13-month-old NSG mice treated twice weekly with saline, PPPF1, or HAS1.Dcx- and Ki67-positive cells were counted in the dentate gyrus of youngand old NSG mice. FIGS. 11A and 11B respectively show that all old micehad dramatically lower numbers of Dcx- or Ki67-positive cells. PPF1 andHAS1-treated mice trended towards increased numbers of Dcx- andKi67-positive cells compared to saline-treated mice.

6. Immunostaining with 3-Month and 13-Month-Old NSG Mice Treated ThreeTimes Weekly with PPF1 and HAS1

Brain sections were stained for doublecortin (Dcx), a marker for newbornneurons or for Ki67, a marker for proliferating cells in 13-month-oldmice. The mice were treated three times per week with saline, PPPF1, 1×concentrated HAS1, or 5× concentrated HAS1. Dcx- and Ki67-positive cellswere counted in the dentate gyrus. FIG. 12 shows that mice treated withPPF1 trended towards an increase in neurogenesis (as indicated by Dcxstaining), compared to saline control treated animals. Also shown isthat more concentrated HAS1 trended towards increased neurogenesiscompared to saline-treated animals.

FIG. 13 shows that mice treated with PPF1 had a significant increase incell proliferation (as indicated by Ki67 staining), compared to salinecontrol treated animals. Also shown is that more concentrated HAS1trended towards increased neurogenesis compared to saline-treatedanimals. *P<0.05; unpaired t-test against saline group; all data shownare mean±s.e.m.

7. Open Field Test with NODscid Mice

NODscid mice were treated twice weekly via intravenous tail veininjection with either saline or PPF1 starting at 6 months of age. Thestarting number of mice were 20 for each group. Mice were placed in theOpen Field chamber for 15 minutes and locomotor activity was recorded.FIG. 14A shows that PPF1-treated mice trend towards increased rearingactivity compared to saline-treated mice. FIGS. 14B and 14C respectivelyshow that PPF1-treated mice also trend towards improved velocity anddistance covered compared to saline-treated mice.

8. Barnes Maze with Aged (12-Month-Old) NSG Mice Treated with YoungPlasma, Effluent I, and Effluent II/III

Aged NSG mice (aged 12 months), were separated into several groups (allof size n=14), and received 150 μL saline, young plasma, Effluent I, orEffluent II/III by tail vein injection prior to initiation of behaviortests. Each separate group was further separated into 3 cohorts witheach cohort initiated with behavior tests on a different week. Mice weretested in a modified Barnes Maze (as described above) to assess spatiallearning and memory. FIG. 15 shows that treatment with young plasma,Effluent I, or Effluent II/III trended towards significant improvementin latency for aged NSG mice to reach the target hole.

9. Barnes Maze and Cell Survival with Aged NSG Mice Treated with YoungPlasma and PPF1

As described above, aged male NSG mice (aged 12 months) were treatedwith 150 μL of clarified young human plasma (young plasma), PPPF1, orsaline three times per week (i.v.) for 4 weeks, and then twice per weekduring weeks 5 and 6, which were the weeks in which testing wasperformed is reported.

FIG. 16 reports the latency to reach the Barnes Maze hole for eachtreatment cohort. Treatment with PPF1 significantly improved spatialmemory in aged mice compared to control, while treatment with youngplasma trended towards improved spatial memory compared to control. (n:Saline=12, PPF1=14, young plasma=11). *P<0.05; mean±s.e.m.; unpairedT-Test.

FIG. 17 reports the average latency to find the target hole for the lastthree trials for each day of testing. Again, treatment with PPF1significantly improved spatial memory in aged mice compared to control,while treatment with young plasma trended towards improved spatialmemory compared to control. *P<0.05; mean±s.e.m.; unpaired T-Test.

FIG. 18 reports the effect of young human plasma and PPF1 on cellsurvival as determined by number of BrdU positively-labeled cells (i.e.proliferating cells) within the granule layer of the dentate gyms ofaged (12 months) NSG mice. BrdU was administered for five days (i.p.)prior to commencing the intravenous injections of young plasma, PPPF1,or saline control as described above. A significant increase in cellsurvival was observed in both young human plasma and PPF1-treated micecompared to saline control. Statistical significance was determinedusing One-Way ANOVA with Dunnett's multiple comparison post-hoc analysisbetween PPF1 and young human plasma compared to saline treatment. (n:Saline=13; PPF1=13; young plasma=11, ****P>0.0001, Unpaired T-Testbetween PPF1 or young human plasma and saline treatment).

L. Results of In Vitro Neurosphere and Cortex Culture Assays

FIG. 19 shows that PPF1 and HAS1 differentially modulate neurosphereproliferation in cortex culture. Cortices from E14-15 C57 mice werecultured on collagen I-coated 96-well plates in culture media containingvehicle alone, PPF1 (10%), or HAS1 (10%). Example images of neurospheresfrom cortical cultures after 21 days in vitro, imaged for Tuj1(neuron-specific class III beta-tubulin), DAPI(4′,6-diamidino-2-phenylindole), or both TuJ1 and DAPI are shown. FIG.19 shows that PPF1 increases the amount of neurospheres which expresseither Tuj1 or DAPI. The increase in Tuj1 expression demonstrates thatPPF1-treated cortical cultures produce more neurospheres which havedifferentiated into a more neuronal-like phenotype.

FIG. 20 depicts three cultures of C57 mouse E14-15 cortical neurons(Lonza: M-CX-300) suspended in neurobasal media supplemented with B27, 2mM Glutamax (Sigma-Aldrich) at 100-200K cells/mL, coated on collagenI-coated 96-well plates in culture media containing vehicle, PPF1 (10%),or HAS1 (10%). Net neurite length, indicative of neurogenesis, occurredin PPF1-treated cultures compared to control or HAS1-treated cultures.

FIG. 21 depicts three cultures of C57 mouse E14-15 cortical neurons(Lonza: M-CX-300) suspended in neurobasal media supplemented with B27, 2mM Glutamax (Sigma-Aldrich) at 100-200K cells/mL, coated on collagenI-coated 96-well plates in culture media containing vehicle, PPF1 (10%),or HAS1 (10%). An IncuCyte software algorithm available from EssenBioSciences (Ann Arbor, Mich.) detected cortex culture spheres(highlighted in yellow) and processes (highlighted in pink). Morespheres and processes were observed in PPF1-treated cultures andincreased sphere size and process branching was also observed inPPF1-treated cultures. The scale bars are 300 μm each.

FIG. 22. FIGS. 22A-D report the number of spheres, the process length,process branch points, and sphere size, respectively. Quantification wasperformed using an IncuCyte software algorithm available from EssenBioSciences (Ann Arbor, Mich.). Standard error is displayed.Significance is shown using a 2-tailed T-Test. FIG. 22A shows thatPPF1-treated cultures have an increased number of spheres compared tovehicle or HAS1-treated cultures. (P=0.0006, PPF1 vs. vehicle; P=0.0007,PPF1 vs. HAS1). FIG. 22B shows that PPF1-treated cultures displayincreased process length compared to vehicle or HAS1-treated cultures.(P=4e⁻⁸, PPF1 vs. vehicle; P=0.002, PPF1 vs. HAS1; and P=0.018, HAS1 vs.vehicle). FIG. 22C shows that PPF1-treated cultures produce more processbranch points compared to vehicle or HAS1-treated cultures. (P=0.002PPF1 vs. vehicle; P=0.004, PPF1 vs. HAS1). FIG. 22D shows thatPPF1-treated cultures are associated with increased sphere size comparedto vehicle or HAS1-treated cultures. (P=0.002 PPF1 vs. vehicle; P=0.004,PPF1 vs. HAS1). Together, the results of this data indicate that PPF1(and HAS1 to a less significant degree) treatment are associated withcharacteristics indicative of increased cortex culture cellular growthand process formation.

FIG. 23 displays the number of neurospheres staining positive for Sox2,a transcription factor which plays an important role in maintainingembryonic and neural stem cells. Quantification was performed using a GEInCell Investigator Toolbox algorithm. PPF1-treated cultures produced asignificantly increased number of neurospheres staining positive forSox2, indicating that PPF1 treatment is associated with an increase innumber of cells with the potential for neurogenesis.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

What is claimed:
 1. A method of treating a cognitive disorder,comprising administering an effective amount of a Plasma Fraction to asubject diagnosed with a cognitive disorder.
 2. The method of claim 1wherein the Plasma Fraction is a Plasma Protein Fraction.
 3. The methodof claim 2 wherein the Plasma Protein Fraction is a commerciallyavailable Plasma Protein Fraction.
 4. The method of claim 1 wherein thePlasma Fraction is a Human Albumin Solution.
 5. The method of claim 4wherein the Human Albumin Solution is a commercially available HumanAlbumin Solution.
 6. The method of claim 1 wherein the Plasma Fractionis a protein-enriched plasma protein product.
 7. The method of claim 1wherein the Plasma Fraction is Effluent I.
 8. The method of claim 1wherein the Plasma Fraction is Effluent II/III.
 9. The method of claim 1further comprising monitoring the subject for improved cognitivefunction.
 10. The method of claim 1 wherein the Plasma Fraction isderived from plasma from a pool of young individuals.
 11. The method ofclaim 1 wherein the Plasma Fraction is produced from a mammalian bloodproduct.
 12. The method of claim 11 wherein the mammalian blood productis a human blood product.
 13. The method of claim 1 wherein the subjectis a mammal.
 14. The method of claim 13 wherein the mammal is a human.15. A kit for use in treating a subject for a cognitive disorder, thekit comprising a containing comprising a Plasma Fraction as described inclaim 1.